Fatty acids as anti-inflammatory agents

ABSTRACT

Compounds of formula I and their metabolites are potent mediators of an inflammatory response:where a, b, c, d, e, f, V, W, X, Y, Ra, Ra′, Rb, Rb′, Rc, and Rc′ are defined herein. In particular, the compounds of the invention are candidate therapeutics for treating inflammatory conditions.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This is a continuation of U.S. application Ser. No. 16/239,425, filedJan. 3, 2019, which is a continuation of U.S. application Ser. No.15/662,024, filed Jul. 27, 2017, issued as U.S. Pat. No. 10,213,417,which is a continuation of U.S. application Ser. No. 14/717,954, filedMay 20, 2015, issued as U.S. Pat. No. 9,750,725, which is a continuationof U.S. application Ser. No. 13/387,489, filed Feb. 8, 2012, issued asU.S. Pat. No. 9,066,902 on Jun. 30, 2015, which is a 371 U.S. NationalStage of International Application No. PCT/US2010/002141, filed on Aug.2, 2010, which application claims priority to U.S. provisionalapplication No. 61/213,946, filed Jul. 31, 2009, the entire contents ofwhich are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support undergrant numbers R01 HL58115 and R01 HL64937, awarded by the NationalInstitutes of Health. The United States government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Electrophilic fatty acids are important transducers of biochemicalinformation. For example, nitro fatty acids mediate cell signalingactivities that are anti-inflammatory in nature. See U.S. PatentPublication No. 20070232579. It is believed that these signaling eventsare regulated by a reversible and covalent modification of thesulfhydryl group of a protein by an electrophilic lipid which results inmodifications of several downstream events such as proteinphosphorylation and activation of transcription to name a few.

Similar to the nitro fatty acids, electrophilic keto fatty acids alsoregulate inflammatory response. The keto fatty acids are generatedduring inflammation due to the up regulation of the enzymecyclooxygenase-2 (COX-2). COX is responsible for formation of importantbiological mediators called prostanoids (including prostaglandins,prostacyclin and thromboxane), of which the prostaglandins are importantpro-inflammatory molecules. Groeger et al. disclose certainelectrophilic fatty acids that are generated during inflammation andthat the corresponding oxo-derivatives were generated by a COX-2catalyzed mechanism in activated macrophages. See Groeger et al.,Cyclooxygenase-2-generates anti-inflammatory mediators from omega-3fatty acids, Nature Chemical Biology 6, 433-441 (2010).

Of the three known COX isoenzymes (COX-1, COX-2 and COX-3),prostaglandins produced by COX-2 are associated pain and inflammation.Thus, agents capable of inhibiting COX-2 have been used as therapeuticsfor treating pain and reducing inflammation.

While selective COX-2 inhibitors are currently known, these compoundsdisplay associated toxic side effects. Thus, their use as therapeuticsin the treatment of chronic pain and inflammation has been limited.

SUMMARY OF THE INVENTION

The present invention provides, in one of its aspects a formulationcomprising (A) a keto fatty acid according to Formula I, and (B) apharmaceutically acceptable carrier.

In Formula (I), X is selected from the group consisting of —CH₂—, —OH,—S, —OR^(t) and —NR^(p)R^(q); Y is selected from the group consisting of—C(O)—, O, —S—, and —NR^(p)R^(q); W is selected from the groupconsisting of —OH, —H, ═S, —SR^(p), —C(O)H, —C(O), —C(O)R^(p), —COOH,—COOR^(p), —Cl, —Br, —I, —F, —CF₃, —CN, —SO₃, —SO₂R^(p), —SO₃H, —NH₃,—NH₂R^(p+), —NR^(p)R^(q)R^(t), NO₂, ═O, ═NR^(p), ═CF₂, and ═CHF and V is—CH— when W is selected from the group consisting of —OH, —H, —C(O)H,—C(O), —C(O)R^(p), —COOH, —COOR^(p), —Cl, —Br, —I, —F, —CF₃, —CN, —SO₃,—SO₂R^(p), —SO₃H, —NH₃+, —NH₂R^(p+), —NR^(p)R^(q)R^(t) and NO₂ and V is—C— when W is selected from the group consisting of ═O, ═NR^(p), ═CF₂,and ═CHF.

The indices a, b, c, d, e, and f independently are integers between 0and 15 inclusive. In one embodiment c is 0 when d is not 0.Alternatively, d is 0 when c is not 0; such that the sums (a+b+c+e+f)and (a+b+d+e+f) independently are equal to an integer that conforms tothe formula 2n or 2n+1, wherein n is an integer between 3 and 15inclusive.

Substituents —R^(p), —R^(q) and —R^(t) are independently selected fromH, (C₁-C₈)alkyl and (C₁-C₈)haloalkyl. In Formula I—R^(a), —R^(a′),—R^(b), —R^(b′), —R^(c), —R^(c′), are independently selected from thegroup consisting of —H, —OH, —C(O)H, —C(O), —C(O)R^(p), —COOH,—COOR^(p), —Cl, —Br, —I, —F, —CF₃, —CHF₂, —CH₂F, —CN, —SO₃, —SO₂R^(p),—SO₃H, —NH₃+, —NH₂R^(p), —NR^(p)R^(q)R^(t) and NO₂. Additionally, —R^(a)and —R^(a′) do not simultaneously represent non-hydrogen groups; —R^(b)and —R^(b′) do not simultaneously represent non-hydrogen groups; and,similarly, —R^(c) and —R^(c′) do not simultaneously representnon-hydrogen groups.

In Formula I, an optional double bond is indicated by

, while

when present, together with X and Y and the carbon atom to which theyare bonded represents a 5- to 6-membered heterocyclyl or heteroarylring. Compounds13-oxo-(7Z,10Z,14A,16Z,19Z)-docosa-7,10,14,16,19-pentanoic acid,17-oxo-(7Z,10Z,13Z,15A,19Z)-docosa-7,10,13,15,19-pentanoic acid, 13-OH(7Z,10Z,14A,16Z,19Z)-docosa-7,10,14,16,19-pentanoic acid, 17-OH(7Z,10Z,13Z,15A,19Z)-docosa-7,10,13,15,19-pentanoic acid,13-oxo-(4Z,7Z,10Z,14A,16Z,19Z)-docosa-4,7,10,14,16,19-hexanoic acid,17-oxo-(4Z,7Z,10Z,13Z,15A,19Z)-docosa-4,7,10,13,15,19-hexanoic acid,13-OH-(4Z,7Z,10Z,14A,16Z,19Z)-docosa-4,7,10,14,16,19-hexanoic acid or17-OH-(4Z,7Z,10Z,13Z,15A,19Z)-docosa-4,7,10,13,15,19-hexanoic acid whereA indicates either E or Z configuration are not covered by Formula I.

In another embodiment, the pharmaceutical formulation comprises a fattyacid selected from the following list13-oxo-(7Z,10Z,14A,16Z,19Z)-docosa-7,10,14,16,19-pentanoic acid,17-oxo-(7Z,10Z,13Z,15A,19Z)-docosa-7,10,13,15,19-pentanoic acid,13-oxo-(4Z,7Z,10Z,14A,16Z,19Z)-docosa-4,7,10,14,16,19-hexanoic acid,17-oxo-(4Z,7Z,10Z,13Z,15A,19Z)-docosa-4,7,10,13,15,19-hexanoic acid,where A indicates either E or Z configuration and a pharmaceuticallyacceptable carrier.

The present invention also provides a method for treating a subjectsuffering from an inflammatory condition comprising administering to thesubject a therapeutically effective amount of a fatty acid according toFormula (I). In another aspect, the invention provides a method fortreating a subject suffering from an inflammatory condition byadministrating a pharmaceutical formulation comprising a fatty acidselected from the group consisting of13-oxo-(7Z,10Z,14A,16Z,19Z)-docosa-7,10,14,16,19-pentanoic acid,17-oxo-(7Z,10Z,13Z,15A,19Z)-docosa-7,10,13,15,19-pentanoic acid, 13-OH(7Z,10Z,14A,16Z,19Z)-docosa-7,10,14,16,19-pentanoic acid, 17-OH(7Z,10Z,13Z,15A,19Z)-docosa-7,10,13,15,19-pentanoic acid,13-oxo-(4Z,7Z,10Z,14A,16Z,19Z)-docosa-4,7,10,14,16,19-hexanoic acid,17-oxo-(4Z,7Z,10Z,13Z,15A,19Z)-docosa-4,7,10,13,15,19-hexanoic acid,13-OH-(4Z,7Z,10Z,14A,16Z,19Z)-docosa-4,7,10,14,16,19-hexanoic acid or17-OH-(4Z,7Z,10Z,13Z,15A,19Z)-docosa-4,7,10,13,15,19-hexanoic acid whereA indicates either E or Z configuration.

Exemplary inflammatory conditions that are treated using the inventiveformulation are organ preservation for transplantation, osteoarthritis,chronic obstructive pulmonary disease (COPD), atherosclerosis,hypertension, allograft rejection pelvic inflammatory disease,ulcerative colitis, Crohn's disease, allergic inflammation in the lung,cachexia, stroke, congestive heart failure, pulmonary fibrosis,hepatitis, glioblastoma, Guillain-Barre Syndrome, systemic lupuserythematosus viral myocarditis, post-transplantation organ protection,acute pancreatitis, irritable bowel disease general inflammation,autoimmune disease, autoinflammatory disease, arterial stenosis, organtransplant rejection and burns, and chronic conditions such as, chroniclung injury and respiratory distress, insulin-dependent diabetes,non-insulin dependent diabetes, hypertension, obesity, arthritis,neurodegenerative disorders, lupus, Lyme's disease, gout, sepsis,hyperthermia, ulcers, enterocolitis, osteoporosis, viral or bacterialinfections, cytomegalovirus, periodontal disease, glomerulonephritis,sarcoidosis, lung disease, lung inflammation, fibrosis of the lung,asthma, acquired respiratory distress syndrome, tobacco induced lungdisease, granuloma formation, fibrosis of the liver, graft vs. hostdisease, postsurgical inflammation, coronary and peripheral vesselrestenosis following angioplasty, stent placement or bypass graft,coronary artery bypass graft (CABG), acute and chronic leukemia, Blymphocyte leukemia, neoplastic diseases, arteriosclerosis,atherosclerosis, myocardial inflammation, psoriasis, immunodeficiency,disseminated intravascular coagulation, systemic sclerosis, amyotrophiclateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer'sdisease, encephalomyelitis, edema, inflammatory bowel disease, hyper IgEsyndrome, cancer metastasis or growth, adoptive immune therapy,reperfusion syndrome, radiation burns, alopecia areta, ischemia,myocardial infarction, arterial stenosis, rheumatoid arthritis, coronaryrestenosis, neurocognitive decline and insulin resistance.

In another embodiment the invention provides a method for detecting ametabolite of a fatty acid according to Formula (I). Detection of one ormore fatty acid metabolites is accomplished by contacting with abiological sample at least one fatty acid according to formula I:

In Formula (I), X is selected from the group consisting of —CH₂—, —OH,—S, —OR^(t) and —NR^(p)R^(q), Y is selected from the group consisting of—C(O)—, O, —S—, and —NR^(p)R^(q), W is selected from the groupconsisting of —OH, —H, ═S, —SR^(p)—C(O)H, —C(O), —C(O)R^(p), —COOH,—COOR^(p), —Cl, —Br, —I, —F, —CF₃, —CN, —SO₃, —SO₂R^(p), —SO₃H, —NH₃ ⁺,—NH₂R^(p+), —NR^(p)R^(q)R^(t), NO₂, ═O, ═NR^(p), ═CF₂, and ═CHF and V is—CH— when W is selected from the group consisting of —OH, —H, —C(O)H,—C(O), —C(O)R^(p), —COOH, —COOR^(p), —Cl, —Br, —I, —F, —CF₃, —CN, —SO₃,—SO₂R^(p), —SO₃H, —NH₃+, —NH₂R^(p+), —NR^(p)R^(q)R^(t) and NO₂ and V is—C— when W is selected from the group consisting of ═O, ═NR^(p), ═CF₂,and ═CHF.

The indices a, b, c, d, e, and f independently are integers between 0and 15 inclusive. In one embodiment c is 0 when d is not 0.Alternatively, d is 0 when c is not 0; such that the sums (a+b+c+e+f)and (a+b+d+e+f) independently are equal to an integer that conforms tothe formula 2n or 2n+1, wherein n is an integer between 3 and 15inclusive.

Substituents —R^(p), —R^(q) and —R^(t) are independently selected fromH, (C₁-C₈)alkyl and (C₁-C₅)haloalkyl. In Formula I —R^(a), —R^(a),R^(a′), —R^(b), —R^(b′), —R^(c), —R^(c′), are independently selectedfrom the group consisting of —H, —OH, —C(O)H, —C(O), —C(O)R^(p), —COOH,—COOR^(p), —Cl, —Br, —I, —F, —CF₃, —CHF₂, —CH₂F, —CN, —SO₃, —SO₂R^(p),—SO₃H, —NH₃ ⁺, —NH₂R^(p+), —NR^(p)R^(q)R^(t) and NO₂. Additionally,—R^(a) and —R^(a′) do not simultaneously represent non-hydrogen groups;—R^(b) and —R^(b′) do not simultaneously represent non-hydrogen groups;and, similarly, —R^(c) and —R^(c′) do not simultaneously representnon-hydrogen groups.

In Formula I, an optional double bond is indicated by

while

when present, together with X and Y and the carbon atom to which theyare bonded represents a 5- to 6-membered heterocyclyl or heteroarylring.

According to the inventive method, a cellular lysate is optionallyprepared depending on the nature of the biological sample. Thebiological sample or cellular lysate is then incubated withβ-mercaptoethanol (BME), for a time sufficient to allow formation of amixture containing one or more covalent BME-fatty acid adducts. Theidentification of one or more fatty acid metabolites is carried out bysubjecting the cellular extract containing BME to mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representative mass spectrum for ions detected uponfragmentation of a BME-keto fatty acid adduct.

FIG. 2 shows the mass of representative ion peaks obtained byfragmentation of a BME-keto fatty acid adduct.

FIG. 3 shows the results from inhibition studies of COX-2. The resultsindicate that formation of electrophilic fatty acid is dependent on thelevel of COX-2.

FIG. 4 is a graphical representation of the results of a fatty acidsupplementation study in cells. It was discovered that the production of22:5 ω-3 keto fatty acid involves a sequence of elongation andde-saturation steps using 18:5 ω-3 fatty acid as the starting material.

FIG. 5 a-5 d illustrates production of EFAD's during macrophageactivation. RAW264.7 cells were activated with PMA (3.24 μM), LPS (0.5μg/ml), and IFNγ (200 U/ml) and harvested 20 h post activation. (a) MRMscans following the neutral loss of 78 (loss of BME) were used to detectelectrophilic fatty acid adducted with BMB in cell extracts fromactivated (a, upper chromatogram) and non-activated (a, lowerchromatogram) RAW 264.7 cells. (b) THP-1 cells were differentiated withPMA (86 nM) for 16 h, activated with Kdo₂ (0.5 μg/ml) and IFNγ (200U/ml), and EFAD levels were detected 8 h post activation. (c) RAW264.7cells were activated with the indicated compounds and EFAD levels werequantified 20 h post activation. Compound concentrations are as follows:LPS (0.5 μg/ml), Kdo₂ (0.5 μg/ml), IFNγ (200 U/ml), PMA (3.24 μM), andfMLP (1 μM). Data are expressed as mean±S.D. (0=4), where*=significantly different (p<0.01) from “PMA+IFNγ+LPS,” and #=asignificant difference (p<0.01) between LPS and “Kdo₂+IFNγ” (one wayANOVA, post-hoc Tukey's test). (d) RA W264.7 cells were activated withKdo2 (0.5 μg/ml) and IFNγ (200 U/ml) and EFAD levels were quantified atindicated times post activation. EFAD-2 levels are reported as generallyrepresentative for other EFADs.

FIG. 6 a-6 e shows that EFAD-2 is an α,β-unsaturated oxo-derivative ofDPA. (a) A characteristic BME electrophile adduct fragmentation pattern.showing the major neutral loss of 78 amu (corresponding to the loss ofBME) is represented by the enhanced product ion analysis of EFAD-2. (b)RAW264.7 cells were grown for 3 days in DMEM and 10% FBS supplementedwith 32 μM of the indicated fatty acid. On the third day cells wereactivated with Kdo₂ (0.5 μg/ml)) and IFNγ (200 U/ml) and EFAD-2 levelswere quantified 20 h post activation. (c) Diagram of NaBH₄ reduction ofoxo-group to an alcohol group. (d) MRM scans monitoring for the mlztransition of 343.2/299.2 (oxo-DPA losing CO₂) in RAW264.7 cell lysatespurified for EFAD-2, non-treated or treated with NaBH₄ (upper and lowerpanel); MRM scans monitoring for the mlz transition of [345.2/327.2(hydroxy-DPA/neutral loss of H20) in RAW264.7 cell lysates purified forEFAD-2 and treated with NaBH₄. (e) MS/MS fragmentation of EFAD-2purified from activated RAW 264.7 cells and reduced with NaBH₄.

FIG. 7 a-7 i shows that EFAD-2 formation is dependent on COX-2 activity.RAW264.7 cells were activated with Kdo₂ (0.5 μg/ml) and IFNγ (200 U/ml)in the presence of the indicated inhibitors and EFAD-2 levels werequantified 20 h post activation. (a) Inhibitor concentrations were asfollows: genistein (25 μM), MAFP (25 μM), MK886 (500 nM), ETYA (25 μM)and OKA (50 nM). (b) COX inhibitor concentrations were as follows: ASA(200 μM), indomethacin (25 μM), ibuprofen (100 μM), diclofenac (1 μM)and NS-398 (4 μM). Data are expressed as mean±S.D. (n=4), where*=significantly different (p<0.01) from “Kdo₂+IFNγ” (one-way ANOVA,post-hoc Tukey's test). (c) The hydroxy-precursors of EFAD-2 weresynthesized using purified ovine COX-2+DPA, ±ASA and quantified (MRM345/327) at the indicated time points. (d, e) Chromatographic profiles(left panels) and spectra (right panels) of the two isomers formed byCOX-2 and COX-2 ASA. (f) RAW264.7 cells were activated with Kdo₂ (0.5μg/ml) and IFNγ (200 U/ml)±ASA and the production of oxoDPA was analyzedand compared to a 17-oxoDPA standard. The elution profile of EFAD-2 wasmonitored by MRM scans following the m/z transition of 421.2/343.2 (theBME adduct of EFAD-2 losing BME). (g-i) RAW264.7 cells were activatedwith Kdo₂ (0.5 μg/ml) and IFNγ (200 U/ml) or treated with vehiclecontrol and lysates were collected 20 h post activation. OH-DPA (μM),DPA (μM), or vehicle was added to the cell lysates and the production ofoxo-DPA or OH-DPA was monitored over time. Full and empty symbolsindicate the use of cell lysates from, respectively, activated andnon-activated cells.

FIG. 8 illustrates formation of EFAD in activated primary murinemacrophages. Bone marrow derived macrophages were activated with Kdo₂(0.5 μg/ml) and IFNγ (200 U/ml) and EFADs were detected 10 h postactivation.

FIG. 9 a-9 b shows the formation of EFAD adducts with proteins in thecell. (a) RAW264.7 cells were activated with Kdo₂ (0.5 μg/ml) and IFNγ(200 U/ml) and harvested 20 h post activation. Cell lysates were thensplit into two groups (and internal standard was added): treatment with500 mM BME followed by protein precipitation with acetonitrile (“Total”)and protein precipitation with acetonitrile followed by treatment with500 mM BME (“Free+small molecule adducted”). EFAD-2 levels werequantified by RP-HPLC-MS/MS. (b) Time-course reaction of EFAD-2 with BMEin RAW264.7 activated cell lysates.

FIG. 10 a-10 b illustrates detection of intracellular and extracellularGS-oxo-DPA adducts following activation of RAW264.7 cells. (a) Chemicalstructure and fragmentation pattern of GS-13-oxoDPA. (b) Chromatographicprofiles and mass spectra of 13- and 17-oxoDPA derived from synthesizedstandards (upper panels), cell medium (middle panel) and cell pellet(lower panel). Differences due to recovery efficiency were taken intoaccount by correcting the signal levels using the internal standardGS-5-oxoETE-d7. Fragments 345.3 and 523.3 were selected and monitored asthe ones giving the best signal to noise ratio in samples derived fromcell media and cell pellets, respectively. Fragment 634.4 derived fromloss of H₂O from the parent ion 652.4; m/z 523.3 and m/z 420.3corresponded to fragments y2 and c1 typical of peptide fragmentationwhile 345.3 and 308.2 derived from the lipid and the glutathionemolecule. m/z 505.3 and m/z 327.2 derived from loss of H₂O from 523.3and 345.3, respectively. K/I, cells treated with Kdo₂ and IFNγ; K/I+ASA,cells treated with Kdo₂, IFNγ and ASA; NT, non-treated cells.

FIG. 11 a-11 e illustrates modulation of anti-oxidant and inflammatoryresponses by 17-oxoDHA and 17-oxoDPA. RAW264.7 cells were treated withincreasing concentration of 17-oxoDHA and 17-oxoDPA. (a) Cells wereharvested 1 h after treatment and Nrf2 levels were quantified in nuclearextracts. (b) Cells were harvested 18 h after treatment and HO-1 andNqo1 (upper band) levels were measured by western blot. (c, d) RAW264.7cells were treated with increasing concentration of 17-oxoDHA and17-oxoDPA for 6 h and Kdo₂+IFNγ were added. Samples were collected at 12h. IL-6, MCP-1 and IL-10 levels were measured in the cell media byQuantikine ELISA Kit (R&D Systems) and normalized by the total proteincontent (c); Nitrite levels were measured in the cell media andnormalized by the total protein content and iNOS and Cox-2 levels weremeasured in total cell lysates (d); (e) PPARγ beta-lactamase reporterassays were performed for Rosiglitazone, 17-OxoDPA, 17-OxoDHA, 15d-PGJ₂,17-hydroxyDHA, DPA, and DHA with concentrations ranging from 0.5-10,000nM.

FIG. 12 illustrates EFADs produced by THP-1 cells coelute with thoseproduced by RAW264.7. THP-1 cells were differentiated with PMA (86 nM)for 16 h, activated with Kdo₂ Lipid A (0.5 μg/ml) and IFNγ (200 U/ml).EFAD levels were detected 8 h post activation. MRM scans following theneutral loss of 78 were used to detect EFAD-BME adducts.

FIG. 13 a-13 c illustrates that BME adducts of the α,β-unsaturatedketo-derivatives yield the most reliable concentration curves forquantification by MS/M.S. (a) The compounds. 9-0xoODE. 12-OxoETE15OxoEDE and 9-OxoOTrE were reacted with BME for 2 h and concentrationcurves were prepared by serial dilution in the presence of 5-OxoETE-d7as internal standard; (b-c) Serial dilution of 9-OxoODE, 12-OxoETE,15-OxoEDE and 9-OxoOTrE were quantified by MRMin the presence ofinternal standard (5-oxoETB-d7) following the neutral loss of CO₂ (b) orby S1M; (c) following parent mass. All peak areas corresponding to thecompounds were normalized to the internal standard and plotted againsttheir concentrations.

FIG. 14 a-f illustrates that EFAD production is dependent on RAW264.7cell activation. RAW24.7 cells were activated with the indicatedcompound and EFAD levels were quantified 20 h post activation. Compoundconcentrations are a follows: LPS (0.5 μg/ml) Kdo₂ Lipid A (0.5 μg/ml)IFNγ (200 U/ml), PMA (3.24 μM). and fMLP (1 μM). Data are expressed asmean±S.D. (n=4), where *=significantly different (p<0.01) from“PMA+IFNγ+LPS,” and #=a significant difference (p<0.01) between LPS and“Kdo₂+IFNγ (one-way ANOVA, post-hoc Tukey s test).

FIG. 15 a-f illustrates that EFAD production is time dependent. RA 264.7cell were activated with Kdo₂ Lipid A (0.5 μg/ml), IFNγ (200 U/ml) andEFAD levels were quantified at indicated times post activation.

FIG. 16 a-16 b illustrates that EFAD-1 and -3 are derived from the n-3series of fatty acids. RAW264.7 cells were grown for 3 days in DMEM and10% FBS supplemented with 32 μM of the indicated fatty acid. On thethird day cells were activated with Kdo₂ Lipid A (0.5 μg/ml) and IFNγ(200 U/ml) and EFAD-1 and -3 levels were quantified 20 h postactivation.

FIG. 17 a-f illustrates that EFAD formation is dependent on PLA₂ andCOX-2 activity. RAW264.7 cells were activated with Kdo₂ Lipid A (0.5μg/ml) and IFNγ (200 U/ml) in the presence of the indicated inhibitorsand EFAD-2. levels were quantified 20 h post activation. Inhibitorconcentrations were as follows: genistein (25 μM), MAFP (25 μM), MK886(500 nM), ETYA (25 μM) and OKA (50 nM). Data are expressed as mean±S.D.(n=4), where *=significantly different (p<0.01) from “Kdo₂+IFNγ”(one-way ANOVA, post-hoc Tukey s test).

FIG. 18 a-f illustrates that EFAD formation is dependent on CO-2activity. RAW264.7 cells were activated with Kdo₂ Lipid A (0.5 μg/ml)and IFNγ (200 U/ml) in th presence of the indicated inhibitors andEFAD-2 levels were quantified 20 h post activation. COX inhibitorconcentrations were as follows: ASA (200 μM), indomethacin (25 μM).ibuprofen. μM), diclofenac (1 μM) and NS-398 (4 μM). Data are expressedas mean±S.D. (n=4), where *=significantly different (p<0.01) from“Kdo₂+IFNγ” (one-way ANOVA, post-hoc Tukey s test).

FIG. 19 a-19 c illustrates that aspirin-acetylated COX-2 produces17-HDHA, rather than 13-HDHA, both in vivo and in vitro. Chromatographicprofiles (a) and: mass spectra (b) of HDHA synthesized by COX-2 inpresence of 10 μM DHA±ASA. Chromatographic profiles of HDHA from celllysates of activated RAW264.7 cells (K/I)±ASA were also compared with13-HDHA and 17-HDHA synthetic standards. (c) Chromatographic:profiles ofoxoDAH generated by activated RAW264.7 cells (K/I)±ASA compared with13-oxoDHA and 17-oxoDHA synthetic standards. Enlarged chromatograms arereported in the insets.

FIG. 20 illustrates that EFADs have a similar reactivity towards BMEcompared to other α,β-unsaturated keto fatty acid. The pseudo firstorder reaction rates of various BME (50 mM) and α,β-unsaturated ketofatty acids (2.9 μM) were measured spectrophotometrically using aAgilent 8453 diode array. The absorbance changes (decrease) werefollowed at 309 nm (15d-PGJ₂), 289 nm (17-oxo-DHA and 17-oxo-DPA) and287 nm (15-oxoETE) (left panel). The reaction was carried out inphosphate buffer pH 7.4 at 37 and 450 spectras were recorded (at 1spectrum per sec) as shown in panels on the right. The decrease inabsorbance was adjusted to a first order-curve using UV-Vis ChemStation(Agilent).

FIG. 21 a-21 h illustrates the mass spectrometric analysis of an invitro reaction of GAPDH with EFAD-2. Four residues were detected andconfined as being targets for EfAD-2 in treated rabbit GAPDH. Thepeptides were alkylated as Cys244 (a), His163 (b), Cys149 (c) and His328(d). Upper panels show EFAD-2 modified peptides and lower panels showspectra from corresponding native peptide.

FIG. 22 illustrates that incubation of EFADs without or with increasingconcentrations of glutathione transferase (GST), resulted in adductionrates that were dependent on the amount of added enzyme confirming thatEFADs were substrate for GSTs.

FIG. 23 illustrates that GS-oxoDHA adducts are detected in pellets andmedia of activated RAW264.7 cells. Chromatographic profiles and massspectra of 13. and 17-oxoDHA derived from synthesized standards (upperpanels) cell medium (middle panel) and cell pellet (lower panel).Differences due to recovery efficiency were taken into account bycorrecting the signal levels using the internal standard GS-5-oxoETE-d7.Fragments 343.3 and 521.3 were selected and monitored as the ones givingthe best signal to noise ratio in samples derived from cell media andcell pellets, respectively. Fragments 521.3 and 418.2 corresponded tofragments y2 and c1, while 343.3 and 308.2 derived from the lipid andthe glutathione molecule. Fragment 503.3 derived from loss of water from523.3.

FIG. 24 a-24 d illustrates that 17-oxoDHA and 7-oxoDPA modulate theinflammatory response. in bone marrow-derived macrophages. Cells weretreated with increasing concentration of 17-oxoDHA and 17oxoDPA for 6 hand Kdo₂ Lipid A (0.5 μg/ml) and IFNγ were added. Samples were collectedat 12 h. (2) Nitrite levels were measured in the cell media andnormalized to the total protein content: iNOS and COX-2 levels weremeasured in total cell lysates. (b) IL-6, MCP-1, and IL-10 levels weremeasured in cell media and normalized to the total protein content.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

The term “alkyl” is used in this description to denote a branched orunbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl,hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyland the like. A “lower alkyl” group is an alkyl group containing fromone to six carbon atoms.

An “alkenyl group” is as a branched or unbranched hydrocarbon group of 2to 24 carbon atoms and structural formula containing at least onecarbon-carbon double bond.

The phrase “alkynyl group” as employed here refers to a branched orunbranched hydrocarbon group of 2 to 24 carbon atoms and containing atleast one carbon-carbon triple bond.

As used herein, “aryl” refers to a monocyclic or polycyclic aromaticgroup, preferably a monocyclic or bicyclic aromatic group, e.g., phenylor naphthyl. Unless otherwise indicated, an aryl group can beunsubstituted or substituted with one or more, and in particular one tofour groups independently selected from, for example, halo, alkyl,alkenyl, OCF₃, NO₂, CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, andheteroaryl. Exemplary aryl groups include but are not limited to phenyl,naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl,trifluoromethylphenyl, nitrophenyl, and 2,4-methoxychlorophenyl.

The term “halogen” and “halo” refers to —F, —Cl, —Br or —I.

The term “heteroatom” is meant to include oxygen (O), nitrogen (N), andsulfur (S).

The term “hydroxyalkyl,” refers to an alkyl group having the indicatednumber of carbon atoms wherein one or more of the alkyl group's hydrogenatoms is replaced with an —OH group. Examples of hydroxyalkyl groupsinclude, but are not limited to, —CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂CH₂H, and branchedversions thereof.

The term “haloalkoyl,” refers to an —(C₁-C₈)alkyl group wherein one ormore hydrogen atoms in the C₁-C₈ alkyl group is replaced with a halogenatom, which can be the same or different. Examples of haloalkyl groupsinclude, but are not limited to, difluoromethyl, trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropylyl, pentachloroethyl,and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “amine or amino” refers to an —NR^(p)R^(q) group wherein RP andR^(q) each independently refer to a hydrogen, (C₁-C₈)alkyl,(C₁-C₅)haloalkyl, and (C₁-C₆)hydroxyalkyl group.

The term “oxo” refers to a ═O atom attached to a saturated orunsaturated (C₃-C₈) cyclic or a (C₁-C₈) acyclic moiety. The ═O atom canbe attached to a carbon, sulfur, and nitrogen atom that is part of thecyclic or acyclic moiety.

The term “heterocycle” refers to a monocyclic, bicyclic, tricyclic, orpolycyclic systems, which are either unsaturated or aromatic and whichcontains from 1 to 4 heteroatoms, independently selected from nitrogen,oxygen and sulfur, wherein the nitrogen and sulfur heteroatoms areoptionally oxidized and the nitrogen heteroatom optionally quaternized,including bicyclic, and tricyclic ring systems. The heterocycle may beattached via any heteroatom or carbon atom. Heterocycles includeheteroaryls as defined above. Representative examples of heterocyclesinclude, but are not limited to, benzoxazolyl, benzisoxazolyl,benzthiazolyl, benzimidazolyl, isoindolyl, indazolyl, benzodiazolyl,benzotriazolyl, benzoxazolyl, benzisoxazolyl, purinyl, indolyl,isoquinolinyl, quinolinyl and quinazolinyl. A heterocycle group can beunsubstituted or optionally substituted with one or more substituents.

“Heterocycloalkyl” denotes to a monocyclic or bicyclic ring systemcontaining one or two saturated or unsaturated rings and containing atleast one nitrogen, oxygen, or sulfur atom in the ring. The term“cycloalkyl” refers to a monocyclic or bicyclic ring system containingone or two saturated or unsaturated rings.

The term “haloalkyl,” refers to a C₁-C₈ alkyl group wherein one or morehydrogen atoms in the C₁-C₆ alkyl group is replaced with a halogen atom,which can be the same or different. Examples of haloalkyl groupsinclude, but are not limited to, trifluoromethyl, 2,2,2-trifluoroethyl,4-chlorobutyl, 3-bromopropyl, pentachloroethyl, and1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroaryl” is employed here to refer to a monocyclic orbicyclic ring system containing one or two aromatic rings and containingat least one nitrogen, oxygen, or sulfur atom in an aromatic ring.Unless otherwise indicated, a heteroaryl group can be unsubstituted orsubstituted with one or more, and preferably one to four, substituentsselected from, for example, halo, alkyl, alkenyl, OCF₃, NO₂, CN, NC, OH,alkoxy, amino, CO₂H, CO₂alkyl, aryl, and heteroaryl. Examples ofheteroaryl groups include, but are not limited to, thienyl, furyl,pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl,triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl,benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

The term n-3, n-6, or n-9 polyunsaturated fatty acids (PUFA); n-3, n-6,or n-9 electrophilic fatty acid derivative (EFAD), respectively; or anyof their respective metabolites is used interchangeably with the termω-3, ω-6, or ω-9 polyunsaturated fatty acids (PUFA), respectively orω-3, ω-6, or ω-9 electrophilic fatty acid derivatives (EFAD),respectively or its metabolites. Similarly, the term omega-3, omega-6,or omega-9 polyunsaturated fatty acids (PUFA), or omega-3, omega-6, oromega-9 electrophilic fatty acid derivatives (EFAD), or its metabolites,refers to the same.

In this context, the category of “metabolites” includes regioisomers,stereoisomers, and structural analogs of keto fatty acids. Thus, theinventive metabolites include fatty acids having tails of differentcarbon length, as well as positional isomers of the double bond. Alsoincluded within the class metabolites are positional isomers of the ketoand hydroxy derivates of PUFA's. Additionally, the double bond can be acis (Z) double bond or a trans (E) double bond. Pursuant to theinvention, moreover, the metabolite category can encompass asmall-molecule analog of a keto fatty acid, as described in greaterdetail below.

The term “derivative” refers to a compound that is derived from asimilar compound, or a compound that can be imagined to arise fromanother compound, if one or more atoms are replaced with another atom orgroup of atoms. Derivatives of the fatty acid metabolites in accordancewith the present invention include without limitation all compounds inwhich one or more carbon atoms in the fatty acid tail are substitutedwith oxygen, sulfur or amino groups. For example, the fatty acid tailcan contain one of more polyethylene glycol units or one or more1,2-diaminoethane units or combinations thereof.

The term “biological sample” refers to tissue, cells, cellular extract,homogenized tissue extract, a mixture of one or more enzymes in asuitable physiologically acceptable carrier, such as a mixture thatincludes without limitation the hydroxy dehydrogenases andcyclooxygenases.

The compound of the invention can also exist in various isomeric forms,including configurational, geometric, and conformational isomers, aswell as existing in various tautomeric forms, particularly those thatdiffer in the point of attachment of a hydrogen atom. The term “isomer”is intended to encompass all isomeric forms of a compound of thisinvention, including tautomeric forms of the compound.

Certain compounds described here may have asymmetric centers andtherefore exist in different enantiomeric and diastereomeric forms. Acompound of the invention can be in the form of an optical isomer or adiastereomer. Accordingly, the invention encompasses compounds of theinvention and their uses as described herein in the form of theiroptical isomers, diastereoisomers and mixtures thereof, including aracemic mixture. Optical isomers of the compounds of the invention canbe obtained by known techniques such as asymmetric synthesis, chiralchromatography, simulated moving bed technology or via chemicalseparation of stereoisomers through the employment of optically activeresolving agents.

Unless otherwise indicated, “stereoisomer” means one stereoisomer of acompound that is substantially free of other stereoisomers of thatcompound. Thus, a stereomerically pure compound having one chiral centerwill be substantially free of the opposite enantiomer of the compound. Astereomerically pure compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, for example greater than about 90%by weight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

If there is a discrepancy between a depicted structure and a name giventhat structure, then the depicted structure controls. Additionally, ifthe stereochemistry of a structure or a portion of a structure is notindicated with, for example, bold or dashed lines, the structure orportion of the structure is to be interpreted as encompassing allstereoisomers of it.

The term “prodrug” denotes a derivative of a compound that canhydrolyze, oxidize, or otherwise react under biological conditions, invitro or in vivo, to provide an active compound, particularly a compoundof the invention. Examples of prodrugs include, but are not limited to,derivatives and metabolites of a compound of the invention that includebiohydrolyzable groups such as biohydrolyzable amides, biohydrolyzableesters, biohydrolyzable carbamates, biohydrolyzable carbonates,biohydrolyzable ureides, and biohydrolyzable phosphate analogues (e.g.,monophosphate, diphosphate or triphosphate). For instance, prodrugs ofcompounds with carboxyl functional groups are the lower alkyl esters ofthe carboxylic acid. The carboxylate esters are conveniently formed byesterifying any of the carboxylic acid moieties present on the molecule.Prodrugs can typically be prepared using well-known methods, such asthose described by BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY6^(th) ed. (Wiley, 2001) and DESIGN AND APPLICATION OF PRODRUGS (HarwoodAcademic Publishers Gmbh, 1985).

The terms “treat”, “treating” and “treatment” refer to the ameliorationor eradication of a disease or symptoms associated with a disease. Incertain embodiments, such terms refer to minimizing the spread orworsening of the disease resulting from the administration of one ormore prophylactic or therapeutic agents to a patient with such adisease.

The term “effective amount” refers to an amount of a compound of theinvention or other active ingredient sufficient to provide a therapeuticor prophylactic benefit in the treatment or prevention of a disease orto delay or minimize symptoms associated with a disease. Further, a“therapeutically effective amount” with respect to a compound of theinvention means that amount of therapeutic agent alone, or incombination with other therapies, that provides a therapeutic benefit inthe treatment or prevention of a disease. Used in connection with acompound of the invention, the term can encompass an amount thatimproves overall therapy, reduces or avoids symptoms or causes ofdisease, or enhances the therapeutic efficacy of or synergies withanother therapeutic agent.

A “patient” or “subject” are used interchangeably throughout thespecification and include an animal (e.g., cow, horse, sheep, pig,chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig), inone embodiment a mammal such as a non-primate and a primate (e.g.,monkey and human), and in another embodiment a human. In one embodiment,a patient is a human. In specific embodiments, the patient is a humaninfant, child, adolescent or adult.

Electrophilic Fatty Acid Derivatives

Polyunsaturated fatty acids exert numerous beneficial health effects inhumans. The major (n-3 PUFAs), are eicosapentanoic acid (EPA;(5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentanoic acid) anddocosahexanoic acid (DHA;(4E,7E,10Z,13E,16E,19E)-docosa-4,7,10,13,16,19-hexanoic acid). Both EPAand DHA exert anti-inflammatory effects by the competitive inhibition ofarachidonic acid-derived prostanoid synthesis and subsequent productionof n-3 prostanoids. In the context of the present invention,electrophilic fatty acid derivatives (EFAD's) are oxidative metabolitesof n-3 PUFAs. Exemplary of an EFAD in accordance with this invention isan α,β-unsaturated keto fatty acid or its metabolites. Keto fatty acidsare lipids in which the ketone group is on a carbon atom adjacent to thecarbon-carbon double bond. Keto fatty acids and their biologicalmetabolites exert biological effects by undergoing adduct formingreactions with nucleophiles present in the biological mileu.

Identification of Keto Fatty Acids and their Metabolites

The present inventors have discovered six electrophilic fatty acidderivatives (EFADs) produced in activated macrophages via a COX-2dependent mechanism. However, acetyl salicylic acid (ASA) treatment ofcells increased the rate of formation and intracellular concentration ofEFAD's. These six EFADs were found at nM concentrations ranging from 65nM to 350 nM in RAW264.7 and were also produced by LPS and IFNγactivated THP-1 cells and primary murine macrophages.

EFADs 1-3 were extensively characterized as α,β-unsaturatedoxo-derivatives of the n-3 fatty acids DHA,(7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentaenoic acid (DPA), anddocosatetraenoic acid (DTA) respectively. Specific 13-oxo and 17-oxopositional isomers have been identified for EFADs 1 and 2, and weresynthesized in vitro. When their biological actions were investigated,EFADs were found to form adducts with proteins and small moleculecellular sulfhydryls, such as GSH, in activated RAW264.7 cells. The17-oxo standards of EFAD-1 and EFAD-2 (17-oxoDHA and 17-oxoDPA) wereable to activate PPARγ and the Keap-1/Nrf2 pathway and to inhibit iNOSand cytokine expression in activated macrophages at concentrations thatparalleled the intracellular concentrations observed in activatedmacrophages.

In one embodiment the present invention describes a class ofenzymatically generated electrophilic fatty acid derivatives (EFADs), ortheir enzymatically generated metabolites. The EFAD's and theirmetabolites have beneficial effects human health. According to theinventors the inventive keto fatty acids or their enzymaticallygenerated metabolites, can inhibit inflammation by giving rise toadaptive signaling molecules in vivo. Since the nature of the responseto an inflammatory condition depends on the cellular levels of aparticular keto fatty acid, the development of an analytical methodcapable of identifying these agents in a biological sample areimportant. The present invention provides a mass spectrometric methodfor analyzing a biological sample. That is, the present method usesβ-mercaptoethanol (BME)-driven alkylation of electrophilic compoundscoupled with reverse phase-high pressure liquid chromatography tandemmass spectrometry (RP-HPLC-MS/MS)²² method for identifying keto fattyacids and their metabolites.

Accordingly, a biological sample of interest is incubated withβ-mercaptoethanol (BME) for a time sufficient to allow a Michaeladdition reaction between β-mercaptoethanol acting as a nucleophile andthe fatty acid metabolites of formulae I, II or III being theelectrophiles, with formation of a mixture containing one or morecovalent β-mercaptoethanol-electrophilic fatty acid adducts. Theidentity of the keto fatty acid in the sample is deduced from theresultant mass peaks in a chromatogram of the sample. By applying thismethod to an in vitro model of inflammation, it was hypothesized thatunknown or poorly characterized species that could be overlooked intraditional screening methods would be more prominently identified. Thealkylation reaction with BME standardizes the MS/MS conditions foradducted RES conferring similar ionization and fragmentation propertieson a range of RES, each with their own particular MS/MS characteristics.Accordingly, reversible RES free or adducted to protein or smallmolecule thiols, species that fragmented poorly during MS/MS, speciesthat were in concentrations at or below the limits of detection, andspecies whose formation was not predictable based on current knowledgecould be identified by this method.

As shown in FIG. 1 , analysis of cellular lysates by the inventivemethod resulted in two peaks that showed a difference in mass of 78daltons, which is attributed to the loss of the BME group (−78 amu;[M-BME]⁻) from the adduct. The net result from such a fragmentation is asecond peak whose mass will correspond to the mass of the keto fattyacid present in cellular lysate. Additional proof confirming theidentity of the keto fatty acid is obtained by fragmenting the M-BMEpeak, followed by analysis of the resultant fragment ions. The advantageof the inventive method is that it provides a good leaving group (i.e.,BME) which enhances the sensitivity and accuracy for the detection of aketo fatty acid in a biological sample.

Using this method six previously uncharacterized major RES speciesformed during activation of RAW 264.7 cells by PMA, LPS, and IFNγ (FIG.5 a ) were identified. MS/MS experiments confirmed the neutral loss ofBME from each of the six EFADS (data not shown). The same electrophilicspecies were detected in PMA, Kdo₂ and IFNγ-activated THP-1 cells, ahuman monocyte/macrophage cell line (FIGS. 5 b and 12). Although theirrelative abundance differed between the two cell lines, MS/MS spectrashowed the same characteristic losses and similar intensity ratios (datanot shown).

The robustness of the BME method for the detection and quantification ofelectrophilic lipids was further tested by comparing the massspectrometric responses of different electrophilic fatty acidscontaining α,β-unsaturated moieties using the BME method, selected ionmonitoring (SIM) and multiple ion monitoring (MRM) mode following theloss of CO₂ (FIG. 13 ). The standard deviation for the overall processesobtained for the different fatty acid at each concentration testedranged from 40% to 50% for BME, 60-83% for MRM and 15-35% for SIManalysis. Moreover, the present inventors have found that the BME basedmass spectrometric methodology was superior to other mass spectrum basedmethods as the former consistently gave a strong signal intensity, a lowbackground level and linearity. The BME method was chosen for biologicalsamples in which SIM and MRM analyses gave very poor results because ofhigh background levels.

EFAD Formation is Time Dependent Following Macrophage Activation.

The formation of EFADs under different inflammatory conditions wasconfirmed by treating the cells with a variety of stimuli. Thus,macrophages were activated with various combinations of LPS, IFNγ, PMA,fMLP, and Kdo₂-Lipid A (Kdo₂) (FIG. 1 e and Supplementary FIG. 3 ).Kdo₂, a synthetic endotoxin, was used to avoid the contribution ofpotential LPS preparation contaminants to EFAD formation. Since thecombination of Kdo₂-Lipid A and IFNγ behaved nearly identically to LPS,it was used for all of the following experiments. This further confirmedthat no components or contaminants in the LPS itself were acting asprecursors of EFADs. Additionally, a time course analysis showed thatEFAD formation started 4-6 h post activation and reached a peak atapproximately 10 h (FIG. 1 d and Supplementary FIG. 4 ). The profiles oftime-dependent formation for the other EFADs were similar to that ofEFAD-2 (data not shown) and the range of intracellular concentrationsobtained for the different species ranged form 65 to 350 nM (Table 1).

EFADs are α,β-Unsaturated Oxo-Derivatives of n-3 Fatty Acids

Confirmation of the presence of an α,β-unsaturated ketone group in EFADinvolved the identification was based on analysis of the mass-time offlight (TOF) data (at an accuracy below 10 ppm), elution profile, andbase on the observance for the loss of CO₂ upon fragmentation of theEFAD. Thus, the EFAD of DPA (EFAD-2), was identified to be amono-oxygenated derivative of a 22-carbon fatty acid tail having a totalof five double bonds. The MS/MS spectrum for BME-adducted EFAD-2 (m/z421 [M-H]⁻) displayed characteristic fragment ions at m/z 403([M-H—H₂O]), 377 ([M-H—CO₂]), 343 ([M-H-BME]), 325 ([M-H-BME-H₂O]), and299 ([M-H-BME-CO₂]) (FIG. 6 a ), consistent with the fragmentationpattern of BME adducts previously reported²².

Similarly, the EFAD of DHA (EFAD-1) and the EFAD of DTA (EFAD-3), wereidentified as mono-oxygenated derivatives of a 22-carbon fatty acid witha total of six and four double bonds, respectively. To elucidate theprecursors in vivo, fatty acid supplementation studies were performed.The formation of EFAD-2 was significantly increased in activatedRAW264.7 cells supplemented with 18:3 n-3 (α-linolenic acid) and 20:5n-3 (EPA) while formation was slightly decreased when the relevant n-6species were provided (FIG. 6 b ). These results indicated that EFAD-2was derived from n-3 PUFAs exclusively. Moreover, as illustrated in FIG.4 EFAD-2 formation is COX-2 dependent. The supplementation of 22:6 n-3(DHA) did not increase EFAD-2 levels. This was consistent with the factthat while mammalian cells can desaturate and elongate shorter chainPUFAs, they generally do not resaturate a PUFA such as DHA.

The formation of EFAD-1 inactivated RAW264.7 cells was increased only bythe supplementation of 22:6 n-3 (FIG. 16 a ). EFAD-3 was increased byboth n-3 and n-6 fatty acid supplementation, indicating that itsprecursor could be either n-3 or n-6 DTA (FIG. 16 b ). Overall, thisstudy showed that EFAD-1, EFAD-2, and a percentage of EFAD-3 werederivatives of n-3 fatty acids DHA, DPA and DTA while EFAD-4 to -6 weresynthesized from n-3 and n-9 fatty acids (Table 1).

TABLE 1 Name EFAD-1 EFAD-2 EFAD-3 EFAD-4 EFAD-5 EFAD-6 Cellularconcentration nM 65 ± 5  238 ± 16  348 ± 26  106 ± 6   326 ± 15  169 ±18  Mass (m/z) 341.2 343.2 345.2 347.2 319.2 321.2 FA precursors 22:618:3n-6, 20:5 20:4, 18:3n-6, 18:3n-6, 20:4 18:3n-6 FBS, 18:1 supportingformation ω-3 ω-3 20:5, 18:3n-3 ω-6 ω-6 ω-9 Series ω-3 and ω-6 Ketogroup 13- or 17- 13- or 17- Yes ? Yes ? present/position positonposition Formula C₂₂H₂₉O₃ C₂₂H₃₁O₃ C₂₂H₃₃O₃ ? C₂₀H₃₁O₃ C₂₀H₃₃O₃ IdentityKeto-DHA Keto-DPA Keto-DTA ω-6 derivative Keto-20:3 Keto-20:2 Activated8.2e⁴ ± 0.55e⁴ 3.3e⁵ ± 0.38e⁵ 2.9e⁵ ± 0.32e⁵ 7.5e⁴ ± 0.53e⁴ 1.7e⁵ ±0.11e⁵ 2.8e⁵ ± 0.24e⁵ (Area/[protein]) ± SD +ETYA (↓COX) ↓ ↓ ↓ ↓ ↓ ↓+Aspirin (↓COX) ↑ ↑ ↑ ↓ ↑ ↓ +Ibuprofen (↓COX) ↓ ↓ ↓ ↓ NE ↓ +Indomethacin(↓COX) ↓ ↓ ↓ ↓ ↓ ↓ +Diclofenac (↓COX) ↓ ↓ ↓ ↓ ↓ ↓ +Celecoxib (↓COX) ↓ ↓↓ ↓ ↓ ↓ +Genisteine ↓ ↓ ↓ ↓ ↓ ↓ +MAFP (↓PLA₂) ↓ ↓ ↓ ↓ ↓ ↓ +MK886(↓5-LOX) NE NE NE N NE NE +OKA NE NE NE NE NE NE

Further confirmation that the electrophilic functional group of EFADswas an α,β-unsaturated carbonyl and to exclude the presence of otherelectrophilic groups (e.g., epoxy group), in the fatty acid tail wasobtained by performing the Luche reaction (FIG. 6 c ). This reactionuses NaBH₄ (in the presence of CeCl₃) to selectively reduce carbonylgroups (but not epoxy or carboxylic acid groups) to the allylic alcoholwithout loss of regioselectivity²³. Lipid extracts from IFNγ andLPS-activated RAW264.7 cell lysates were fractionated by HPLC. Thefraction containing EFAD-2 was purified and reduced with NaBH₄ resultingin a significant decrease of the signal corresponding to EFAD-2 and theappearance of a previously absent peak at the transition 345/327(reduced product of EFAD-2, FIG. 6 d ). Due to the enhancedfragmentation typically induced by hydroxy-groups during MS/MS, productsof the Luche reaction yielded relevant information about the location ofthe carbonyl group. Accordingly, in addition to the commonly observedion fragments (m/z 327 ([M-H]—H₂O) and 283 ([M-H]—H₂O—CO₂), thefollowing diagnostic ions for 13-hydroxy-DPA (13-OH-DPA) were observedin the EFAD-2 enriched fraction reduced with NaBH₄: m/z 223, 205(223-H₂O) and 195 (FIG. 6 e ). These findings finally revealed thatEFAD-2 corresponded to 13-oxoDPA and that EFAD-3 was an oxo-derivativeof DTA. (Table 1)

The BME technique is useful to identify the biologically importantmetabolites of electrophilic keto fatty acids. For example, the BMEtechnique was used by the inventors to fish out the biologically activeC-10 to C₁₈ metabolites which are potent anti-inflammatory electrophilicsignaling molecules. The small, low molecular weight metabolites haveimproved stability and bio-availability, making these compounds as wellas derivatives of these fatty acid metabolites candidate therapeuticsfor the treatment chronic pain and inflammation.

Keto Fatty Acids and their Metabolites

The major n-3 polyunsaturated fatty acids (n-3 PUFAs) eicosapentanoicacid (EPA; (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentanoic acid) anddocosahexanoic acid (DHA;(4E,7E,10Z,13E,16E,19E)-docosa-4,7,10,13,16,19-hexanoic acid) have beenassociated with numerous beneficial health effects in humans. Inparticular, brain and retina tissues are enriched with DHA in healthyindividuals and DHA is necessary for the normal development and functionof these tissues^(1,2). Moreover, the consumption of DHA in the diet hasbeen implicated in reducing neurocognitive decline³, improving insulinresistance in diabetics⁴, decreasing incidence of cardiovascular riskssuch as myocardial infarction⁵, and reducing inflammation⁶. Both EPA andDHA exert anti-inflammatory effects by competitive inhibition ofarachidonic acid-derived prostanoid synthesis, and subsequent productionof n-3 prostanoids with the ability to induce vasodilation, inhibitplatelet aggregation⁷ and promote a series of anti-inflammatory eventswhose mechanisms remain to be elucidated.

Several emerging classes of anti-inflammatory lipid mediators have beenrecently reported. Although structurally related to pro-inflammatoryprostanoids, these lipid derivatives promote resolution of inflammationby suppressing NF-κB activation, modulating cytokine expression,activating G-protein coupled receptors⁸ and promoting cyto-protectiveresponses⁹. Among these are the enzymatically synthesized resolvins(Rvs), neuroprotectins, maresins, and lipoxins (LXs). Oxygenases,including cyclooxygenase-2 (COX-2) and lipoxygenases (LOXs), areinvolved in these biosynthetic processes emerging as key enzymes both inthe onset of inflammatory events and in their finely orchestratedresolution¹⁰.

A second group of such lipid derivatives include nitro-fatty acids(NO₂—FAs), 15-deoxy-Δ (12,14)-prostaglandin J₂ (15d-PGJ₂), andneuroprostanes which are reactive electrophilic species (RES) mostlyformed during non-enzymatic oxidative events. In the context of thisinvention, electrophilic fatty acid derivatives (EFAD's), are oxidativemetabolites of n-3 PUFAs. Exemplary of an EFAD is a keto fatty acid.Keto fatty acids are lipids in which there is a ketone group adjacent tothe carbon atoms of the double bond. Keto fatty acids as well as theirbiological metabolites are reactive electrophilic species (RES), thatexert their biological effects mainly via electrophilicreactions^(11,12).

RES are molecules characterized by having an electron-withdrawingfunctional group that renders the α-carbon electron-poor and reactivetowards electron-rich donor molecules (nucleophiles). The strength ofthe electron withdrawing group will determine the reactivity of theelectrophile. Exemplary of an electron withdrawing group present in theinventive keto fatty acids or their metabolites is an α,β-unsaturatedcarbonyl group which can undergo a Michael addition reaction withbiological nucleophiles. A more detailed characterization of RES ispresented below herein.

Thus, when inflammation is initiated in mouse cells (RAW264.7) and humanmonocyte cells (THP-1), respectively, intracellular levels of keto fattyacid are elevated. In particular, six electrophilic fatty acidderivatives (EFADs) were identified in activated macrophages by thepresent inventors. These EFADS in activated macrophages werecharacterized by mass spectrometry as α,β-unsaturated oxo-derivatives ofthe n-3 fatty acids DHA,(7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentanoic acid (DPA), anddocosatetranoic acid (DTA) respectively. Specifically 13-oxo and 17-oxopositional isomers have been identified for the DHA and DPA EFADs.

Biological Production of EFAD

1. Role of COX-2

Not wishing to be bound by any particular theory, the present inventorsbelieve that the EFAD's are produced in activated cells via a COX-2dependent mechanism. For example, in the process of identifying EFADs asα,β-unsaturated oxo-derivatives of PUFAs, a series of experiments wereperformed to determine the pathways involved in their synthesis. EFADlevels were quantified in RAW264.7 cells activated with Kdo₂ and IFNγand treated with a variety of inhibitors (FIG. 7 a , FIG. 17 , and Table1).

Both genistein and methyl arachidonyl fluorophosphonate (MAFP) inhibitedEFAD production by over 50%. Genistein was chosen as a general tyrosinekinase inhibitor to inhibit LPS and IFNγ signal transduction. MAFP, aselective irreversible inhibitor of both calcium-dependent andcalcium-independent cytosolic phospholipase A2 (cPLA₂ and iPLA₂), wasemployed to determine if EFAD precursors were released from thecytoplasmic membrane upon RAW264.7 cell activation. To determine if5-lipoxygenase (5-LOX) was involved in EFAD formation, MK886 was used toprevent FLAP-dependent activation of 5-LOX. MK886 had no significanteffect on EFAD.

Eicosatetraynoic acid (ETYA), a nonspecific inhibitor of COX and LOXenzymes, was found to strongly inhibit EFAD formation, while the generalphosphatase inhibitor, okadaic acid (OKA), caused a slight increase inEFAD formation, probably due to the enhancement of LPS and IFNγ signaltransduction. In order to determine whether the inhibitory effect ofETYA on EFAD formation was due specifically to the inhibition of COX,EFAD levels were quantified in RAW264.7 cells that were activated withKdo₂ and IFNγ, and treated with COX inhibitors at concentrations thatwere at least 5 times their IC₅₀ values²⁴ (FIGS. 7 c , and 18, and Table1).

Indomethacin and diclofenac were found to completely abolish EFADformation, while ibuprofen was found to significantly inhibit EFADformation by more than 80%, with the exception of EFAD-1, which showedno significant effect. Moreover, the selective COX-2 inhibitor NS-398, aclose structural relative of Nimesulide, abolished EFAD formation aswell. That is, COX-2 specific conventional non-steroidalanti-inflammatory drugs (NSAIDs) used to reduce inflammation also reducethe resultant levels of cellular keto fatty acids. See FIG. 3 .

Finally, acetyl salicylic acid (ASA), significantly increased EFADformation by about 2.5 fold for all EFADs, with the only exception beingEFAD-4 and EFAD-6. This was consistent with previous reports showingthat ASA acetylation of COX-2 Ser530 favors the formation ofmono-oxygenated derivatives of long chain PUFAs²⁵. A summary of theresults obtained with the inhibitor study for all EFADs is reported inTable 1.

The results of the COX-2 inhibition study implicate a the involvement ofCOX-2 in EFAD formation and motivated the development of an in vitromodel of enzymatic EFAD synthesis. Purified ovine COX-2 was used togenerate the EFAD-2 precursor (OH-DPA), (FIG. 7 c-e ), while the EFAD-1precursor, hydroxy-DHA (OH-DHA), was produced from DHA by COX-2 (FIGS.19 a and 19 b ). Interestingly, ASA increased the rate and extent offormation of OH-DPA (FIG. 7 c ) and shifted the population ofhydroxy-isomers produced from 13- to 17- (FIG. 7 d-f and FIGS. 19 a and19 b ).

Analysis of the enzymatic reaction mixture using mass spectrometryshowed a characteristic fragmentation pattern. For the fragmentationpattern of COX-derived 13-OH-DPA characteristic m/z 195 and 223 ionswere observed, which ions correspond to the hydroxyl group inducedfragmentation observed for RAW264.7 cell extracts that were subjected toa reduction reaction using sodium borohydride (NaBH₄) (FIGS. 6 e and 7 d). In contrast, when COX-2 reaction mixture was treated with ASA,characteristic ions corresponding to a hydroxyl group at C-17 positionwere detected (FIG. 7 e ). In activated, ASA-treated RAW264.7 cells thisshift resulted in the production of 17-oxo-isomers, as shown in FIG. 7 gfor EFAD-2 and FIG. 19 c for EFAD-1.

2. Role of Hydroxydehydrogenases

The conversion of PUFA's to their corresponding oxo-derivatives requiresthe presence of hydrodehydrogenases in addition to COX-2. For example,lysates from activated or non-activated RAW264.7 cells were incubatedwith the EFAD-2 precursors DPA and OH-DPA. When activated andnon-activated cell lysates were incubated with OH-DPA in presence ofNAD⁺ there was a time-dependent production of EFAD-2 (FIG. 7 g ). Incontrast, only lysate from activated cells displayed a time-dependentproduction of OH-DPA and EFAD-2 when incubated with DPA (FIG. 7 g-i ).

These results show that only activated cells are able to metabolicallyconvert DPA into its oxo derivative (oxo-DPA), confirming the role ofCOX-2 in the conversion of PUFA's to their corresponding hydroxyderivatives which are converted to the corresponding oxo derivativesenzymatically via hydroxy-dehydrogenases that appear to beconstitutively expressed. According to the present inventors, therefore,a linear correlation exists between the in vivo levels of a keto fattyacid and the in vivo level of COX-2. The formation of EFAD's in othercell lines such as primary cell lines was also determined by the presentinventors as further explained below in the experimental section.

EFADs Activate Cyto-Protective and Anti-Inflammatory Pathways

As described above, compounds of the invention can react with biologicalthiols to form reversible covalent adducts. Thus, intracellular RES'ssuch as EFAD's promote the activation of the Nrf2-dependent anti-oxidantresponse pathway via thiol-dependent modification of the Nrf2 inhibitorKeap1. This induces nuclear translocation of the transcription factorNrf2 and the expression of its target genes²⁸. For example, the17-oxoDHA and 17-oxoDPA promoted dose-dependent Nrf2 nuclearaccumulation and expression of the cytoprotective enzymes heme oxygenase1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (Nqo1) (FIGS. 11 a and 11b ).

To investigate whether the 17-oxo DHA and 17-oxo DPA played a role inmodulating the inflammatory response generated by Kdo₂ and IFNγ, thepresent investigators study the change in levels of cytokines, IL-6,MCP-1 and IL-10 in cells exposed to increasing doses of 17-oxo DHA and17-oxo DPA.

The intracellular levels of both MCP-1 and IL-10 were depressed in adose-dependent manner following EFAD treatment. For example, ˜80%reduction in the levels of MCP-1 was observed at the highestconcentration of EFADs, while approximately 50% reduction was observedIL-6 (FIG. 11 c ). Similar results were observed in bone marrow-derivedmacrophage (BMDMs), (FIG. 23 a ). As shown in FIG. 11 d , EFAD-1 and -2strongly and dose-dependently repressed inducible nitric oxide synthase(iNOS) induction and subsequent accumulation of nitrite in the cellmedia both in RAW264.7 and in BMDMs (FIG. 23 b ).

In particular, a ˜70% reduction of nitrite production was observed at17-oxoDPA and 17-oxoDHA concentration of 25 and 20 μM respectively.Interestingly, Cox-2 induction was not affected by EFADs in this study.The expression of iNOS and the analyzed pro-inflammatory cytokines isdependent on the activity of NF-κB and Stat-1. It has been reported thatelectrophilic lipids can repress the activation of these transcriptionalfactors either by direct adduction to the DNA binding domain of theNF-κB subunit p65 and to the inhibitor IκBα or via indirect mechanisms.However, EFADs do not significantly inhibit p65 nuclear translocationand DNA binding or Stat phosphorylation.

The observation that oxo-fatty acids, such as 15d-PGJ₂ ³⁰, 5-oxoEPA,6-oxoOTE, and the synthetic 4-oxoDHA³¹, covalently bind and activate theperoxisome proliferator-activated receptor γ (PPARγ), prompted thepresent inventors to test the ability of 17-oxoDPA, and 17-oxoDHA toactivate PPARγ. Thus PPARγbeta-lactamase reporter assays were performedusing Roziglitazone, a potent synthetic PPARγ agonist, was used in theassay as positive control.

Both EFAd's (17-oxoDPA and 17-oxoDHA), activated PPARγ (FIG. 11 e ) withslightly higher EC₅₀s (˜40 nM) as compared to the natural ligand15d-PGJ₂ (˜25 nM) and EC₅₀s that were orders of magnitude lower than17-OH-DPA (>10 M) and their corresponding native fatty acids (DHA andDPA).

Because, of their reactivity with biological thiols, the inventiveEFAD's and their metabolites are believed to react with a cysteine,present in the binding pocket of the transcription factor of the COX-2gene, and thereby inactivate the transcription factor. The result is aninhibition of inflammation by a lowering of cellular COX-2 levels.

The present invention also provides certain mimetics of Keto fatty acidsor their metabolites whose synthesis is as described below.

Synthesis of the Mimetics of Keto Fatty Acid

The α,β-unsaturated ketone unit, found in a many bioactive molecules, isan important synthon in medicinal chemistry. Several syntheses have beenreported for bioactive molecules involving the α,β-unsaturated ketoneunit. See Synder, B. et al., Org Lett., (2001), 3(4), 569-572 andBamford, S. et al., Org Lett., (2000), 2(8), 1157-1160.

Against this background, the inventors found that fatty acids accordingto Formula I are potent mediators of inflammatory response.

In particular, α,β-unsaturated keto fatty acids show a stronganti-inflammatory effect. Without endorsing any particular theory, theinventors believe that, in some embodiments, the keto and carboxylategroups of the unsaturated lipid contribute to electrostatic and hydrogenbonding interactions with residues that line the binding pocket of aneffector protein.

The present invention, therefore, provides keto fatty acid mimetics thatretain the above mentioned electrostatic and hydrogen bondinginteractions. In some embodiments of the invention, mimetics thatconform to Formula II below, are analogs of a heterocyclic dioneconjugated to an α,β-unsaturated alkyl ketone. The dione functionalityof the inventive mimetic is believed to occupy the same region withinthe effector protein's binding pocket as does the carboxylate head groupof the lipid. Thus, the dione functionality would interact with theprotein in the manner of the carboxylate group of a biological ketofatty acid. Moreover, by maintaining the position of the keto group inthe tail region of the mimetic, compounds Formula (II) are believed tobind tightly to their targets.

The synthesis of compounds shown in Formula II can be achieved byreacting an appropriately substituted nitrile with diethyl phosphonate,followed by a base catalyzed reaction of the enamine phosphonate with analdehyde. Scheme 1 depicts this synthetic strategy.

This strategy is versatile, allowing the conjugation of differentheterocyclic diones to an appropriately functionalized α,β-unsaturatedalkyl ketone. Thus, X in Scheme 1 can be a sulfur, oxygen or anunsubstituted or appropriately substituted nitrogen atom.

The strategy depicted in Scheme 1 also allows for the synthesis ofmimetics that bear a substituent group on the carbon alpha to the ketogroup. Thus, R₁ in Scheme 1 is selected from the group consisting ofhydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₄-C₈)bicycloalkyl, (C₃-C₈)heterocycloalkyl,heteroaryl, aryl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl andaryl(C₁-C₆)alkyl.

In a further embodiment, the inventive mimetic is a triazole derivative.See Formula III. Several studies indicate that the triazole unit is amimic of a carboxylate group. Thus, putative mimetics incorporating atriazole unit in place of the carboxylate moiety are believed to bind ina manner similar to keto fatty acids to physiological targets implicatedin anti-inflammatory activity. These compounds therefore are candidatetherapeutics for treating inflammation.

Compounds shown by Formula III can readily be synthesized using “click”chemistry. Thus, reaction of the azide of an α,β-unsaturated alkylketone with appropriately substituted alkynes in the presence of acatalyst results in the triazole mimetic. Scheme 2 illustrates thevarious synthetic steps that lead to the inventive triazole mimetics.

Thus, R₁, R₂ and R₃ in Scheme 2 are each independently selected from thegroup consisting of hydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, (C₁-C₄)alkoxy, (C₁-C₄)alkoxy(C₁-C₄)alkyl,(C₁-C₅)fluoroalkyl, (C₁-C₈)hydroxyalkyl, (C₃-C₈)cycloalkyl,(C₄-C₈)bicycloalkyl, (C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl and aryl(C₁-C₆)alkyl.

Formulations of Keto Fatty Acids, Metabolites and Mimetics

In accordance with one of its aspects, the present invention provides aformulation of a keto fatty acid, its metabolite or mimetic that comportwith Formulae I-III, and their pharmaceutically acceptable salt, solvateor hydrate and a pharmaceutically acceptable carrier. Also contemplatedare formulations having one or more therapeutic agents in addition tocompounds of the invention. Non-limiting examples of therapeutics addedto the inventive formulation include chemotherapeutic agents,antibodies, antivirals, steroidal and non-steroidal anti-inflammatories,conventional immunotherapeutic agents, cytokines, chemokines, and/orgrowth factors. In a further aspect, the inventive composition containstwo or more of the Formulae I-III compounds described above, formulatedtogether.

In a formulation of the invention, more than one physiologicallyacceptable carrier can be used, such as a mixture of two or morecarriers. Additionally, an inventive formulation can include thickeners,diluents, solvents, buffers, preservatives, surface active agents,excipients, and the like.

The compounds of the invention can include pharmaceutically acceptablecations include metallic ions and organic ions. More preferred metallicions include, but are not limited to, appropriate alkali metal salts,alkaline earth metal salts and other physiological acceptable metalions. Exemplary ions include aluminum, calcium, lithium, magnesium,potassium, sodium and zinc in their usual valences. Preferred organicions include protonated tertiary amines and quaternary ammonium cations,including in part, trimethylamine, diethylamine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Exemplarypharmaceutically acceptable acids include, without limitation,hydrochloric acid, hydroiodic acid, hydrobromic acid, phosphoric acid,sulfuric acid, methanesulfonic acid, acetic acid, formic acid, tartaricacid, maleic acid, malic acid, citric acid, isocitric acid, succinicacid, lactic acid, gluconic acid, glucuronic acid, pyruvic acid,oxalacetic acid, fumaric acid, propionic acid, aspartic acid, glutamicacid, benzoic acid, and the like.

Isomeric and tautomeric forms of inventive compounds of the invention aswell as pharmaceutically acceptable salts of these compounds are alsoencompassed by the invention. Exemplary pharmaceutically acceptablesalts are prepared from formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic,stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic,cyclohexylaminosulfonic, algenic, beta.-hydroxybutyric, galactaric andgalacturonic acids.

Suitable pharmaceutically acceptable base addition salts used inconnection with the inventive compounds of the invention includemetallic ion salts and organic ion salts. Exemplary metallic ion saltsinclude, but are not limited to, appropriate alkali metal (group Ia)salts, alkaline earth metal (group Ia) salts and other physiologicalacceptable metal ions. Such salts can be made from the ions of aluminum,calcium, lithium, magnesium, potassium, sodium and zinc. Preferredorganic salts can be made from tertiary amines and quaternary ammoniumsalts, including in part, trimethylamine, diethylamine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. All of theabove salts can be prepared by those skilled in the art by conventionalmeans from the corresponding compound of the present invention.

Pharmaceutical formulations containing the compounds of the inventionand a suitable carrier can be in various forms including, but notlimited to, solids, solutions, powders, fluid emulsions, fluidsuspensions, semi-solids, and dry powders including an effective amountof an inventive compound of the invention. It is also known in the artthat the active ingredients can be contained in such formulations withpharmaceutically acceptable diluents, fillers, disintegrants, binders,lubricants, surfactants, hydrophobic vehicles, water soluble vehicles,emulsifiers, buffers, humectants, moisturizers, solubilizers,antioxidants, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, Marcel Deldcer, Inc. (1979); and Goodman& Gilman's, The Pharmaceutical Basis of Therapeutics, 6th Edition,MacMillan Publishing Co., New York (1980) both of which are herebyincorporated by reference in their entireties can be consulted.

The compounds of the present invention can be formulated for parenteralor intravenous administration by injection, e.g., by bolus injection orcontinuous infusion. Formulations for injection can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids diluents such as oleic acid finduse in the preparation of injectables. Additional fatty acids diluentsthat may be useful in embodiments of the invention include, for example,one or more of stearic acid, metallic stearate, sodium stearyl fumarate,fatty acid, fatty alcohol, fatty acid ester, glyceryl behenate, mineraloil, vegetable oil, paraffin, leucine, silica, silicic acid, talc,propylene glycol fatty acid ester, polyethoxylated castor oil,polyethylene glycol, polypropylene glycol, polyalkylene glycol,polyoxyethylene-glycerol fatty ester, polyoxyethylene fatty alcoholether, polyethoxylated sterol, polyethoxylated castor oil,polyethoxylated vegetable oil, and the like. In some embodiments, thefatty acid diluent may be a mixture of fatty acids. In some embodiments,the fatty acid may be a fatty acid ester, a sugar ester of fatty acid, aglyceride of fatty acid, or an ethoxylated fatty acid ester, and inother embodiments, the fatty acid diluent may be a fatty alcohol suchas, for example, stearyl alcohol, lauryl alcohol, palmityl alcohol,palmitolyl acid, cetyl alcohol, capryl alcohol, caprylyl alcohol, oleylalcohol, linolenyl alcohol, arachidonic alcohol, behenyl alcohol,isobehenyl alcohol, selachyl alcohol, chimyl alcohol, and linoleylalcohol and the like and mixtures thereof.

In other embodiments the inventive formulations are solid dosage formsfor oral administration including capsules, tablets, pills, powders, andgranules. In such embodiments, the active compound may be admixed withone or more inert diluent such as sucrose, lactose, or starch. Suchdosage forms may also comprise, as in normal practice, additionalsubstances other than inert diluents, e.g., lubricating agents such asmagnesium stearate. In the case of capsules, tablets, and pills, thedosage forms may also comprise buffering agents and can additionally beprepared with enteric coatings.

The solid dosage form can be a liquid or gelatin formulation prepared bycombining the inventive compound with one or more fatty acid diluent,such as those described above, and adding a thickening agent to theliquid mixture to form a gelatin. The gelatin may then be encapsulatedin unit dosage form to form a capsule. In another exemplary embodiment,an oily preparation of an inventive compound prepared as described abovemay be lyophilized to for a solid that may be mixed with one or morepharmaceutically acceptable excipient, carrier or diluent to form atablet, and in yet another embodiment, the inventive compound of an oilypreparation may be crystallized to from a solid which may be combinedwith a pharmaceutically acceptable excipient, carrier or diluent to forma tablet.

Further embodiments which may be useful for oral administration ofinventive compounds include liquid dosage forms. In such embodiments, aliquid dosage may include a pharmaceutically acceptable emulsion,solution, suspension, syrup, and elixir containing inert diluentscommonly used in the art, such as water. Such compositions may alsocomprise adjuvants, such as wetting agents, emulsifying and suspendingagents, and sweetening, flavoring, and perfuming agents.

In still further embodiments, inventive compounds of the invention canbe formulated as a depot preparation. Such long acting formulations canbe administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Depot injections can beadministered at about 1 to about 6 months or longer intervals. Thus, forexample, the compounds can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

Other suitable diluents for injectable formulations include, but are notlimited to those described below:

Vegetable oil: As used herein, the term “vegetable oil” refers to acompound, or mixture of compounds, formed from ethoxylation of vegetableoil, wherein at least one chain of polyethylene glycol is covalentlybound to the vegetable oil. In some embodiments, the fatty acids hasbetween about twelve carbons to about eighteen carbons. In someembodiments, the amount of ethoxylation can vary from about 2 to about200, about 5 to 100, about 1.degree. to about 80, about 20 to about 60,or about 12 to about 18 of ethylene glycol repeat units. The vegetableoil may be hydrogenated or unhydrogenated. Suitable vegetable oilsinclude, but are not limited to castor oil, hydrogenated castor oil,sesame oil, corn oil, peanut oil, olive oil, sunflower oil, saffloweroil, soybean oil, benzyl benzoate, sesame oil, cottonseed oil, and palmoil. Other suitable vegetable oils include commercially availablesynthetic oils such as, but not limited to, Miglyol™ 810 and 812(available from Dynamit Nobel Chemicals, Sweden) Neobee™ M5 (availablefrom Drew Chemical Corp.), Alofine™ (available from Jarchem Industries),the Lubritab™ series (available from JRS Pharma), the Sterotex™(available from Abitec Corp.), Softisan™ 154 (available from Sasol),Croduret™ (available from Croda), Fancol™ (available from the FanningCorp.), Cutina™ HR (available from Cognis), Simulsol™ (available from CJPetrow), EmCon™ CO (available from Amisol Co.), Lipvol™ CO, SES, andHS-K (available from Lipo), and Sterotex™ HM (available from AbitecCorp.). Other suitable vegetable oils, including sesame, castor, corn,and cottonseed oils, include those listed in R. C. Rowe and P. J.Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which isincorporated herein by reference in its entirety. Suitablepolyethoxylated vegetable oils, include but are not limited to,Cremaphor™ EL or RH series (available from BASF), Emulphor™ EL-719(available from Stepan products), and Emulphor™ EL-620P (available fromGAF).

Mineral oils: As used herein, the term “mineral oil” refers to bothunrefined and refined (light) mineral oil. Suitable mineral oilsinclude, but are not limited to, the Avatech™ grades (available fromAvatar Corp.), Drakeol™ grades (available from Penreco), Sirius™ grades(available from Shell), and the Citation™ grades (available from AvaterCorp.).

Castor oils: As used herein, the term “castor oil”, refers to a compoundformed from the ethoxylation of castor oil, wherein at least one chainof polyethylene glycol is covalently bound to the castor oil. The castoroil may be hydrogenated or unhydrogenated. Synonyms for polyethoxylatedcastor oil include, but are not limited to polyoxyl castor oil,hydrogenated polyoxyl castor oil, mcrogolglyceroli ricinoleas,macrogolglyceroli hydroxystearas, polyoxyl 35 castor oil, and polyoxyl40 hydrogenated castor oil. Suitable polyethoxylated castor oilsinclude, but are not limited to, the Nikkol™ HCO series (available fromNikko Chemicals Co. Ltd.), such as Nikkol HCO-30, HC-40, HC-50, andHC-60 (polyethylene glycol-30 hydrogenated castor oil, polyethyleneglycol-40 hydrogenated castor oil, polyethylene glycol-50 hydrogenatedcastor oil, and polyethylene glycol-60 hydrogenated castor oil,Emulphor™ EL-719 (castor oil 40 mole-ethoxylate, available from StepanProducts), the Cremophore™ series (available from BASF), which includesCremophore RH40, RH60, and EL35 (polyethylene glycol-40 hydrogenatedcastor oil, polyethylene glycol-60 hydrogenated castor oil, andpolyethylene glycol-35 hydrogenated castor oil, respectively), and theEmulgin® RO and HRE series (available from Cognis PharmaLine). Othersuitable polyoxyethylene castor oil derivatives include those listed inR. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients,(2006), 5th ed., which is incorporated herein by reference in itsentirety.

Sterol: As used herein, the term “sterol” refers to a compound, ormixture of compounds, derived from the ethoxylation of sterol molecule.Suitable polyethoyxlated sterols include, but are not limited to, PEG-24cholesterol ether, Solulamm C-24 (available from Amerchol); PEG-30cholestanol, Nikkol™ DHC (available from Nikko); Phytosterol, GENEROL™series (available from Henkel); PEG-25 phyto sterol, Nikkol™ BPSH-25(available from Nikko); PEG-5 soya sterol, Nikkol™ BPS-5 (available fromNikko); PEG-10 soya sterol, Nikkol™ BPS-10 (available from Nikko);PEG-20 soya sterol, Nikkol™ BPS-20 (available from Nikko); and PEG-30soya sterol, Nikkol™ BPS-30 (available from Nikko). As used herein, theterm “PEG” refers to polyethylene glycol.

Polyethylene glycol: As used herein, the term “polyethylene glycol” or“PEG” refers to a polymer containing ethylene glycol monomer units offormula —O—CH.sub.2-CH.sub.2-. Suitable polyethylene glycols may have afree hydroxyl group at each end of the polymer molecule, or may have oneor more hydroxyl groups etherified with a lower alkyl, e.g., a methylgroup. Also suitable are derivatives of polyethylene glycols havingesterifiable carboxy groups. Polyethylene glycols useful in the presentinvention can be polymers of any chain length or molecular weight, andcan include branching. In some embodiments, the average molecular weightof the polyethylene glycol is from about 200 to about 9000. In someembodiments, the average molecular weight of the polyethylene glycol isfrom about 200 to about 5000. In some embodiments, the average molecularweight of the polyethylene glycol is from about 200 to about 900. Insome embodiments, the average molecular weight of the polyethyleneglycol is about 400. Suitable polyethylene glycols include, but are notlimited to polyethylene glycol-200, polyethylene glycol-300,polyethylene glycol-400, polyethylene glycol-600, and polyethyleneglycol-900. The number following the dash in the name refers to theaverage molecular weight of the polymer. In some embodiments, thepolyethylene glycol is polyethylene glycol-400. Suitable polyethyleneglycols include, but are not limited to the Carbowax™ and Carbowax™Sentry series (available from Dow), the Lipoxol™ series (available fromBrenntag), the Lutrol™ series (available from BASF), and the Pluriol™series (available from BASF).

Propylene glycol fatty acid ester: As used herein, the term “propyleneglycol fatty acid ester” refers to an monoether or diester, or mixturesthereof, formed between propylene glycol or polypropylene glycol and afatty acid. Fatty acids that are useful for deriving propylene glycolfatty alcohol ethers include, but are not limited to, those definedherein. In some embodiments, the monoester or diester is derived frompropylene glycol. In some embodiments, the monoester or diester hasabout 1 to about 200 oxypropylene units. In some embodiments, thepolypropylene glycol portion of the molecule has about 2 to about 100oxypropylene units. In some embodiments, the monoester or diester hasabout 4 to about 50 oxypropylene units. In some embodiments, themonoester or diester has about 4 to about 30 oxypropylene units.Suitable propylene glycol fatty acid esters include, but are not limitedto, propylene glycol laurates: Lauroglycol™ FCC and 90 (available fromGattefosse); propylene glycol caprylates: Capryol™ PGMC and 90(available from Gatefosse); and propylene glycol dicaprylocaprates:Labrafac™ PG (available from Gatefosse).

Stearoyl macrogol glyceride: Stearoyl macrogol glyceride refers to apolyglycolized glyceride synthesized predominately from stearic acid orfrom compounds derived predominately from stearic acid, although otherfatty acids or compounds derived from other fatty acids may used in thesynthesis as well. Suitable stearoyl macrogol glycerides include, butare not limited to, Gelucire® 50/13 (available from Gattefosse).

In some embodiments, the diluent component comprises one or more ofmannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powderedcellulose, microcrystalline cellulose, carboxymethylcellulose,carboxyethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodiumstarch glycolate, pregelatinized starch, a calcium phosphate, a metalcarbonate, a metal oxide, or a metal aluminosilicate.

Exemplary excipients or carriers for use in solid and/or liquid dosageforms include, but are not limited to sorbitols such as PharmSorbidexE420 (available from Cargill), Liponic 70-NC and 76-NC (available fromLipo Chemical), Neosorb (available from Roquette), Partech SI (availablefrom Merck), and Sorbogem (available from SPI Polyols).

Starch, sodium starch glycolate, and pregelatinized starch include, butare not limited to, those described in R. C. Rowe and P. J. Shesky,Handbook of Pharmaceutical Excipients, (2006), 5th ed., which isincorporated herein by reference in its entirety.

Disintegrant: The disintegrant may include one or more of croscarmellosesodium, carmellose calcium, crospovidone, alginic acid, sodium alginate,potassium alginate, calcium alginate, an ion exchange resin, aneffervescent system based on food acids and an alkaline carbonatecomponent, clay, talc, starch, pregelatinized starch, sodium starchglycolate, cellulose floc, carboxymethylcellulose,hydroxypropylcellulose, calcium silicate, a metal carbonate, sodiumbicarbonate, calcium citrate, or calcium phosphate.

Still further embodiments of the invention include inventive compoundsadministered in combination with other active such as, for example,adjuvants, protease inhibitors, or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein.

Route of Administration

The inventive compounds of the invention can be administered in anyconventional manner by any route where they are active. Administrationcan be systemic or local. For example, administration can be, but is notlimited to, parenteral, subcutaneous, intravenous, intramuscular,intraperitoneal, transdermal, oral, buccal, or ocular routes, orintravaginally, by inhalation, by depot injections, or by implants. Incertain embodiments, the administration may be parenteral orintravenous, all in the presence or absence of stabilizing additivesthat favor extended systemic uptake, tissue half-life and intracellulardelivery. Thus, modes of administration for the compounds of the presentinvention (either alone or in combination with other pharmaceuticals)can be injectable (including short-acting, depot, implant and pelletforms injected subcutaneously or intramuscularly). In some embodiments,an injectable formulation including an inventive compound may bedeposited to a site of injury or inflammation, such as, for example, thesite of a surgical incision or a site of inflammation due toarthroscopy, angioplasty, stent placement, by-pass surgery and so on.

In certain other embodiments, the compounds of the invention may beapplied locally as a salve or lotion applied directly to an area ofinflammation. For example, in some embodiments, a lotion or salveincluding inventive compounds of the invention may be prepared andapplied to a burn, radiation burn, site of dermal disorder, edema,arthritic joint or the like.

Various embodiments, of the invention are also directed to method foradministering inventive compounds. Specific modes of administration mayvary and may depend on the indication. The selection of the specificroute of administration and the dose regimen may be adjusted or titratedby the clinician according to methods known to the clinician in order toobtain the optimal clinical response. The amount of compound to beadministered is that amount which is therapeutically effective. Thedosage to be administered will depend on the characteristics of thesubject being treated, e.g., the particular animal treated, age, weight,health, types of concurrent treatment, if any, and frequency oftreatments, and can be easily determined by one of skill in the art(e.g., by the clinician). Those skilled in the art will appreciate thatdosages may be determined with guidance, for example, from Goodman &Goldman's The Pharmacological Basis of Therapeutics, Ninth Edition(1996), Appendix II, pp. 1707-1711 or from Goodman & Goldman's ThePharmacological Basis of Therapeutics, Tenth Edition (2001), AppendixII, pp. 475-493 both of which are hereby incorporated by reference intheir entireties.

In various embodiments, an effective amount of an inventive compounddelivered during each administration cycle may range from about 10mg/m.sup.2/day to about 1000 mg/m²/day. In some embodiments, aneffective amount may be about 20 mg/m²/day to about 700 mg/m²/day, andin others, an effective amount may be about 30 mg/m²/day to about 600mg/m²/day. In particular embodiments, an effective amount may be about50 mg/m²/day, about 400 mg/m²/day, about 500 mg/m²/day, or about 600mg/m²/day. In yet other embodiments, an effective amount of an inventivecompound may vary as treatment progresses. For example, a dosage regimenmay be increased or decreased as treatment proceeds throughadministration cycles, or the daily dosage may increase or decreasethroughout administration. In additional embodiments, greater than 1000mg/m²/day may be administered because even high doses of inventivecompound are generally tolerable to the patient and may not produceundesired physiological effects.

The pharmaceutical carrier used to formulate the inventive compoundswill depend on the route of administration. Administration may betopical (including opthamalic, vaginal, rectal, or intranasal), oral, byinhalation, or parenterally, for example by intravenous drip,subcutaneous, intraperitoneal or intramuscular injection.

Thus, the compounds of the invention can be administered intravenously,intraperitoneally, intramuscularly, subcutaneously, intracavity,transdermally, intratracheally, extracorporeally, or topically (e.g.,topical intranasal administration or administration by inhalant). Inthis regard, the phrase “topical intranasal administration” connotesdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. The latter can be effective when a large number ofsubjects are to be treated simultaneously. Administration of thecompositions by inhalant can be through the nose or mouth via deliveryby a spray or droplet mechanism. Delivery can also be directed to anyarea of the respiratory system (e.g., lungs) via intubation.

Formulations of the inventive keto fatty acid mimetics for parenteraladministration will include excipients and carriers that stabilize thenitro fatty acid mimetic. Illustrative of such a carrier are non-aqueoussolvents, such as propylene glycol, polyethylene glycol, vegetable oils,and injectable organic esters such as ethyl oleate. Additionally,formulations for parenteral administration include liquid solutions,suspensions, or solid forms suitable for solution or suspension inliquid prior to injection, or emulsions.

Intravenous formulations of the mimetics include agents to maintain theosmomolarity of the formulation. Examples of such agents include sodiumchloride solution, Ringer's dextrose, dextrose, lactated Ringer'ssolution, fluid and nutrient replenishers, and the like. Also includedin intravenous formulations are one or more additional ingredients thatprevent microbial infection or inflammation, as well as anesthetics.

The present invention also provides formulations of the pharmaceuticallyacceptable salts of the inventive mimetics. Illustrative of such saltsare those formed by reaction of the mimetics with an inorganic base suchas sodium hydroxide, ammonium hydroxide, or potassium hydroxide. Alsocontemplated are salts of the inventive mimetics with organic bases suchas mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

In yet another aspect, a mimetic of the invention can be formulated as aprodrug. At physiological pH, a mimetic of a keto fatty acid typicallywill be a charged molecule, which may have non-optimal bioavailabilityand cell-transport kinetics. To address these concerns, therefore, onemay provide a compound of the invention as a pharmaceutically acceptableester, such as a methyl or an ethyl ester. The ester acts as a prodrugbecause non-specific intracellular esterase convert it in vivo to theactive form.

Methods of Treatment

Compounds in accordance with the present invention may be administeredto individuals to treat, ameliorate and/or prevent a number both acuteand chronic inflammatory and metabolic conditions. In particularcompounds in accordance with Formulae I-III as well as their metabolitesmay be used to treat acute conditions including general inflammation,autoimmune disease, autoinflammatory disease, arterial stenosis, organtransplant rejection and burns, and chronic conditions such as, chroniclung injury and respiratory distress, diabetes, hypertension, obesity,arthritis, neurodegenerative disorders and various skin disorders.However, in other embodiments, inventive compounds may be used to treatany condition having symptoms including chronic or acute inflammation,such as, for example, arthritis, lupus, Lyme's disease, gout, sepsis,hyperthermia, ulcers, enterocolitis, osteoporosis, viral or bacterialinfections, cytomegalovirus, periodontal disease, glomerulonephritis,sarcoidosis, lung disease, lung inflammation, fibrosis of the lung,asthma, acquired respiratory distress syndrome, tobacco induced lungdisease, granuloma formation, fibrosis of the liver, graft vs. hostdisease, postsurgical inflammation, coronary and peripheral vesselrestenosis following angioplasty, stent placement or bypass graft,coronary artery bypass graft (CABG), acute and chronic leukemia, Blymphocyte leukemia, neoplastic diseases, arteriosclerosis,atherosclerosis, myocardial inflammation, psoriasis, immunodeficiency,disseminated intravascular coagulation, systemic sclerosis, amyotrophiclateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer'sdisease, encephalomyelitis, edema, inflammatory bowel disease, hyper IgEsyndrome, cancer metastasis or growth, adoptive immune therapy,reperfusion syndrome, radiation burns, alopecia and the like.

When administered inventive compounds may interact with a number ofcellular receptors and/or proteins that mediate inflammation, either byinhibiting or stimulating their activity thereby inhibiting or reducinginflammation. Without wishing to be bound by theory, the inventorsbelieve that inventive compounds can modulate important signalingactivities including, for example, neurotransmission, gene expression,vascular function and inflammatory responses. Chemical properties ofinventive compounds that may facilitate these activities include, butare not limited to, the strong, reversible electrophilic nature of theβ-carbon adjacent to the electron withdrawing vinyl group, an ability toundergo Nef-like acid base reactions to release NO, an ability topartition into both hydrophobic and hydrophilic compartments, and astrong affinity for G-protein coupled receptors and nuclear receptors.

For example, in one embodiment, the inventive compounds may beadministered to mediate cell signaling via multiple G-protein coupledreceptors and nuclear receptors such as, but not limited to, peroxisomeproliferator-activated receptors (PPAR) including PPAR.alpha.,PPAR.gamma., and PPAR.delta. PPAR is a nuclear receptor that isexpressed throughout an organism, including in monocytes/macrophages,neutrophils, endothelial cells, adipocytes, epithelial cells,hepatocytes, mesangial cells, vascular smooth muscle cells, neuronalcells and when “activated” induces transcription of a number of targetgenes. Activation of PPAR has been shown to play various roles inregulating tissue homeostasis including, for example, increasing insulinsensitivity, suppress chronic inflammatory processes, reduce circulatingfree fatty acid levels, correct endothelial dysfunction, reduce fattystreak formation, delay plaque formation, limit blood vessel wallthickening and enhance plaque stabilization and regression. Theinventive compounds embodied herein may perform each of these functionsassociated with PPAR activation.

Moreover, inventive compounds may perform these functions withoutsignificantly altering normal cellular process. For example, in oneembodiment, an inventive compound may be administered to treathypertension by lowering blood pressure to normal levels withoutreducing the blood pressure of the individual below normal levels evenif the inventive compound is over-administered. Thus, without wishing tobe bound by theory, the compounds of the invention may provide treatmentof an individual without the negative affects associated withover-administration or over-treatment using traditional medications.

EXAMPLES Experimental Methods

Materials

Diclofenac, methyl arachidonyl fluorophosphonate, MK886, (±)-Ibuprofen,Indomethacin, NS-398, 15d-PGJ₂, 4-hydroxy-2-nonenal, 9-OxoODE,5-OxoETEd7, 12-OxoETE, 15-OxoEDE, 9-OxoOTrE, 17-OxoDPA, and 17-OxoDHAwere purchased from Cayman Chemicals (Ann Arbor, Mich.). Ovine placentalCOX-2 (Cayman 60120) was also from Cayman Chemicals. DPA and DHA werefrom NuCheck Prep (Elysian, Minn.). Kdo₂ lipid A was from Avanti PolarLipids, Inc (Alabaster, Ala.). HPLC solvents were from Honeywell Burdickand Jackson (USA). Glutathione and glutathione S-transferase werepurchased from Sigma-Aldrich.

Cell Culture and Treatment

Murine monocyte/macrophage cells (RAW264.7) and human monocyte cells(THP-1) were obtained from ATCC (USA) and maintained at 37° C. in 5% CO₂in DMEM+10% FBS (RAW264.7) and RPMI+10% FBS (THP-1) according to ATCCguidelines. L-cells were obtained from ATCC (CCL-1) and maintained at37° C. in 5% CO₂ in DMEM supplemented with 10% FBS, glutamine (2 mM),sodium pyruvate (1 mM), penicillin, streptomyocin and non-essentialamino acids.

For activation experiments RAW 264.7 cells were seeded, incubatedovernight, and treated at approximately 80% confluence with theindicated compounds 47. Non-activated controls were treated with vehiclealone. During activation, cells were maintained in an activation medium(SMEM) of Minimum Essential Medium Eagle (Cellgro, 17-305)+2% FBSsupplemented with L-glutamine (584 mg/L), Na-pyruvate (110 mg/L) andHepes (3.57 g/L, pH 7.4). For inhibition studies, inhibitors were addedto the medium at the time of activation and MTT assays were used toconfirm cell viability. Cells were harvested 20 h post activation(unless otherwise indicated) in 50 mM phosphate buffer (pH 7.4) and snapfrozen in liquid N₂. THP-1 cells were differentiated with PMA (86 nM)for 16 h, activated with IFNγ (200 U/ml) and Kdo2 lipid A (0.5 μg/ml) inRPMI+2% FBS and harvested 40 h after differentiation. For treatment withEFADs alone, 17-oxoDHA and 17-oxoDPA were added to cell culture media atthe indicated concentrations and for the indicated time period. Fortreatment with EFADs coupled with pro-inflammatory stimulation, additionof 17-oxoDHA and 17-oxoDPA was followed by addition of Kdo₂ and IFNγ at6 h.

Trans-Alkylation Reaction of Electrophiles with BME.

Upon thawing, lysates were exposed to BME (500 mM+internal standard,5-OxoETEd7 (1.25 ng/ml)) and incubated at 37° C. for 1 h in 50 mMphosphate buffer (pH=7.4) as previously described²². Proteins wereprecipitated with cold acetonitrile and the supernatant was analyzed byHPLC-ESI-MS/MS.

HPLC-ESI-MS/MS

Samples were separated by reverse-phase HPLC using a 20×2 mm C18 MercuryMS column (3 μm, Phenomenex). A gradient solvent system was usedconsisting of A (water/0.1% formic acid) and B (acetonitrile/0.1% formicacid) at 750 μl/min under the following conditions: hold at 35% B for0.5 min, then 35-90% B in 4 min, 90-100% B in 0.1 min, hold for 1.4 minand 100-35% B for 0.1 min, hold for 1.9 min. To achieve resolution ofisomers, chromatographic runs were performed using a 150×2 mm C18 Lunacolumn (3 μm, Phenomenex). A flow rate of 250 μl/min was used under thefollowing conditions: hold at 35% B for 3 min, then 35-90% B for 23 min,then 90-100% B in 0.1 min, hold for 5.9 min and 100-35% B for 0.1 min,hold for 7.9 min. The analysis and quantification of BME adducts wereperformed using a hybrid triple quadrupole-linear ion trap massspectrometer (4000 Q trap, Applied Biosystems/MDS Sciex) in the neutralloss (NL) scan mode, multiple reaction monitoring (MRM) scan mode, andthe enhanced product ion analysis (EPI) mode. The following settingswere used: declustering potential −90 and −50 V, and collision energy−30 and −17 V for free fatty acids and BME adducts, respectively. Zerograde air was used as source gas, and N₂ was used in the collisionchamber. EFADs were quantified using external synthetic standards, whenavailable, and by comparing peak area ratios between analytes and a5-OxoETEd7 internal standard. Data were acquired and analyzed usingAnalyst 1.4.2 software (Applied Biosystems, Framingham, Mass.).

COX-2 Reactions

Ovine placental COX-2 (20 U/ml) was preincubated in Tris/heme/phenol(THP) buffer±2 mM ASA at 37° C. THP buffer, freshly prepared before eachreaction, consisted of Tris.Cl (100 mM, pH 8.1), hematin (1 μM), andphenol (1 μM). The reaction was initiated by addition of the indicatedfatty acids at a concentration of 10 μM. Reactions were terminated atthe indicated time points by addition of ice-cold acetonitrile(9×reaction volume) and COX-2 protein was removed by centrifugation.Product formation was monitored by RP-HPLC-MS/MS in multiple reactionmonitoring (MRM) mode following the loss of CO₂ (m/z 345/301 and m/z343/299 for OH-DPA and OH-DHA, respectively).

Preparation of Primary Macrophages

Bone marrow derived macrophages were isolated from C57BL/6 miceaccording to the protocol developed by Davies. See Davies, J. Q. &Gordon, S. Isolation and culture of murine macrophages. Methods Mol Biol290, 91-103 (2005).

Western Blot

Protein concentrations of samples were measured by BCA assay (Pierce).The following primary antibodies were used: Nrf2 (Santa Cruz, sc-722),HO-1 (Assay Design, SPA-896), Nqo1 (Abcam, ab34173), Cox-2 (Santa Cruz,sc-1745), iNOS (BD Transduction Lab, 610332), Lamin B1 (Abcam, ab16048).Actin (detected by Sigma A2066) was used as loading control. Secondaryantibodies were purchased from Santa Cruz Biotechnology.

Nitrate/Nitrite Measurement

Total nitrite and nitrate concentration was measured in cell culturemedia by Griess reaction using the Nitrate/Nitrite Colorimetric AssayKit (Cayman Chemical).

Measurement of Glutathione Adducts

GS-adducts were analyzed in cell pellets and media by nano-LC-MS/MSusing nanoACQUITY UltraPerformance LC coupled with Thermo-Fisher LTQ.GS-5-oxoETE-d7 was added as internal standard. Waters XBridge BEH130 C18NanoEase Column (3.5 μm, 100 μm×100 mm) was used. Chromatography wasperformed using a binary flow system consisting of A (H₂O/0.1% formicacid) and B (acetonitrile/0.1% formic acid) at 0.5 μl/min under thefollowing conditions: hold at 1.5% B for 3 min, then 1.5 to 30% B in 10min, then 30 to 70% B in 27 min. The following parent ions weremonitored for identification of, respectively, GS-5-oxoETE-d7, GS-oxoDHAand GS-oxoDPA: 633.3, 650.3 and 652.3.

Statistics.

Data are expressed as mean±SD and were evaluated by a one-way analysisof variance, post-hoc Tukey's test for multiple pairwise comparisons.Significance was determined as p<0.01 unless otherwise indicated.

Reactive Electrophilic Species (RES)

RES are molecules characterized by having an electron-withdrawingfunctional group that renders the α-carbon electron-poor and reactivetowards electron-rich donor molecules (nucleophiles). The strength ofthe electron withdrawing group will determine the reactivity of theelectrophile. Two prominent examples of these electron withdrawinggroups are β,β-unsaturated carbonyls and nitroalkenes, in which theβ-carbon (if it is bound to at least one hydrogen atom) is the site ofnucleophilic attack. The resonance structure of electrophiles like theseallows them to react covalently with many nucleophiles via Michaeladdition. Interestingly, the reactivity of the electrophilic compoundappears to directly relate to the biological outcome of eachelectrophile¹³ with irreversible adducts conveying toxic effects¹⁴. Inaddition, RES also modulate the cell redox potential by changing theGSH/GSSG redox couple, which can further impact underlying cellsignaling. By covalently modifying proteins, RES can initiate cellsignaling events and modulate enzymatic activity and subcellularlocalization¹⁵. RES production and levels are tightly controlled inhealthy cells with low levels of these species inducing the expressionof cell survival genes, and in some cases priming the cells to surviveperiods of stress. In contrast, under pathological conditions, RES areoften produced in excess and overcome signaling events and protectivepathways, accelerating cell damage¹⁶. Recently, there has been a movetowards employing RES in the prevention or treatment of various diseasessuch as neurodegeneration, cancer, and other pathologies presenting asignificant inflammatory component. For example, electrophilic neuriteoutgrowth-promoting prostaglandin compounds display protective effectsduring cerebral ischemia/reperfusion, which are attributed to theiraccumulation in neurons and subsequent activation of the Keap1/Nrf2pathway¹⁷. Other RES (e.g. avicins¹⁸ andBis(2-hydroxybenzylidene)acetone¹⁹, isothiocyanates²⁰) are potentialchemopreventative agents, due to their abilities to induce apoptosis ofprecancerous cells and tumor cells. Additionally, the electrophile15d-PGJ₂ demonstrates a protective role in animal models of acute lunginjury²¹.

EFADs are Produced by Primary Macrophages Isolated from Mouse BoneMarrow

Since RAW 264.7 cells (and potentially other macrophage cell lines) havean altered AA metabolism²⁶, it was important to demonstrate that theformation of EFADs occurred in primary cell lines as well. Thus, C57BL/6murine primary hematopoietic stem cells were differentiated tomacrophages, activated with Kdo₂ and IFNγ and analyzed for the formationof EFADs. Five out of the six EFAD species (EFAD-1, 2, -3, -5 and -6)were observed which co-eluted with those produced by RAW 264.7 cells andwith the available standards. Similar to what was observed in RAW264.7cells, when activated bone marrow-derived macrophage (BMDM) cells weretreated with ASA the extent of EFAD formation was increased about two tothree fold and in the case of EFAD-1 and -2, the isomeric compositionshifted from 13-oxo to 17-oxo species (FIG. 8 ).

Kinetics and Identification of EFADs Adduct to Proteins and Glutathione(GSH)

Biological electrophiles, such as EFAD's react with sulfhydryl groups ofproteins as well as the cellular reductant GSH^(15,27-29). Differentapproaches have been used to demonstrate the occurrence and extent ofadduct formation by EFAD's to proteins and small molecule sulfhydryls.To demonstrate the occurrence of sulfhydryl adducts in activated cells,total EFAD content was quantified and compared with the pool of freeEFADs (including EFADs adducted to small molecules such as glutathione).The difference between the two groups gave the percentage of EFADsadducted to proteins (˜51%) (FIG. 9 a ). To confirm the distribution ofintracellular EFADs, between free and adduct form both free and adductedEFAd's were allowed to react with BME. The difference in reactionkinetics of free EFAD with BME and adducted EFAD with BME was used toconfirm the distribution of intracellular EFADs, between free and adductform.

Typically, reaction rates of BME with free electrophiles is fast with acalculated pseudo first order reaction rate constant in the range ofabout 3×10⁻³ and 5×10⁻³ sec⁻¹ for the different α,β-unsaturatedoxo-fatty acids tested (15d-PGJ₂, EFAD-1, EFAD-2) (FIG. 20 ). Incontrast, reactions rates with adducted electrophiles are slower,depending on the rate constant (k_(off)), for the Cys-EFAD and His-EFADadducts. The time-dependent characteristic of these reactions was usedto further confirm the adducted populations present in the cell lysates(FIG. 9 b ).

Fast kinetics with free EFADs and a slower t reaction rate for thedisplacement of EFADs from adducted proteins were observed.Approximately 50% of the EFADs reacted with BME within the first 5 min,suggesting that protein-adducted EFADs accounted for the remaining ˜50%of total EFADs that reacted with BME after 45 min (FIG. 9 b ).

To more specifically test the binding of EFADs to nucleophilic residuesin proteins, we tested whether GAPDH was alkylated by EFADs. This enzymeis a well-characterized target for electrophiles and becomes easilyinactivated by nitrosylation, oxidation or nucleophilic addition. Asexpected and based on its electrophilic properties, the EFAD-2 syntheticstandard (17-oxo-isoform) readily formed adducts with Cys244, Cys149,His163 and His328 residues of GAPDH in vitro (FIG. 21 ).

The cellular reductant glutathione reacts readily with biologicalelectrophiles, such as EFAd's, via the sulhydryl group of cysteine togive the corresponding glutathione-EFAD (GS-EFAD), adduct. The presentinventors investigated whether EFADs were substrates for glutathioneS-transferase (GST) and if GS-EFADs adducts were actually formed incells during macrophage activation.

Thus, incubation of EFADs without or with increasing concentrations ofGST resulted in adduction rates that were dependent on the amount ofadded enzyme confirming that EFADs were substrate for GSTs (FIG. 22 ).

FIGS. 10 a, 10 b and 22 illustrates results of a mass spectral analysisof glutathione adducts from cell lysates, cell medium of activatedRAW264.7 cells, and compares these mass spectrums to the mass spectrumobtained from a reaction mixtures of a synthetically prepared GS-oxoDHAand GS-oxoDPA standard. The fragmentation patterns and retention timesobserved for GSH adducts of EFAD-1 and -2 corresponded to those obtainedusing the synthetic standards. Moreover, the addition of ASA enhancedthe formation of GS-adducts, consistent with the concomitant increase inEFAD synthesis. GS-adducts were also found in the extracellular media,the only exception being that for samples treated with ASA, detection ofGS-adducts in the extracellular media was unexpectedly reduced.

Discussion

Despite current knowledge on a wide range of lipid signaling mediators,the question as posed by Harkewicz et al. still remains: “arebiologically significant eicosanoids [or other fatty acid-derivedmetabolites] being overlooked?” Herein we address this question byfocusing the search for negatively charged lipid metabolites on thosewith reversible electrophilic activity and consequently potentialsignaling capabilities. The methods used in this study detected sixnovel EFADs, as well as oxoETE (data not shown), that were produced byactivated macrophages. To the best of our knowledge, five of thesespecies have not been described before as relevant mediators ofinflammation or as metabolic products formed by mammalian cells (EFAD-5may correspond to oxoETrE). Interestingly, 15d-PGJ₂ was not observed inthis study; the levels of 15d-PGJ₂ may have been too low for detection,implying that the novel species reported here may also be responsiblefor the effects often attributed to 15d-PGJ₂.

In taking the search for lipid mediators a step further, the LipidMetabolites and Pathway Strategy consortium (Lipid MAPS;http://www.lipidmaps.org), has been publishing information focused onthe lipid section of the metabolome and “global changes in lipidmetabolites” (i.e. lipidomics) since 2005. While the methods used todate have identified new lipid metabolites and yielded valuable data onthe signaling properties of these metabolites, they have theirlimitations and the potential to overlook lipids with unique orunconventional means of signal transduction. Other studies use methodsthat have focused exclusively on RES; by using MS/MS to detect and studyRES-GSH adducts, it is possible to appreciate the in vivo signature leftby various RES and to obtain structural information on RES of interestby using MS³. However, there are also limitations in using this method.For example, RES generated in lipid bilayers may not have theopportunity to interact with GSH, but may still modify membraneassociated proteins. This concept has already been used to characterizeenzyme-generated RES produced by the hypersensitive response in tobaccoleaves.

The inventors have developed an alternative to analyzing only RES-GSHadducts, in which an alkylation reaction of electrophiles toβ-mercaptoethanol (BME) is used to identify electrophiles that canreversibly adduct to cellular sulfhydryls (or other nucleophiles).Conventionally, oxidized PUFA species have been discovered byhypothesizing the substrates, mechanisms/enzymes, and subsequentlyidentifying the products of labeled substrates or identifying thehypothesized products, as compared to synthetic compounds. The successof this method is exemplified by the extensive knowledge of various PGspecies and the discovery of isoprostanes, neuroprostanes, lipoxins andResolvins. Conversely, the oxidized lipid species reported in this studywere initially discovered exclusively based on their chemicalproperties: negatively charged small hydrophobic molecules withreversible electrophilic activity. The BME method used herein increasedMS/MS sensitivity for RES and standardized the behavior of a variety ofRES during MS/MS analysis. For example, oxo-fatty acid derivatives donot fragment as well as the corresponding hydroxy-derivatives, renderingstructural identification more difficult. Accordingly, one reason thatthe species described in this work have not been reported before may bethat the typical method of lipid metabolite identification yieldslargely the expected or the most abundant species; unanticipated lipidspecies that might be produced and signal at lower concentrations wouldbe relegated to the background of more prominent species in this method.In the present work we report previously uncharacterized electrophilicfatty acids, which were primarily derived by oxygenation of n-3 PUFAs.In particular, EFAD-1 to -3 corresponded to oxoDHA, oxoDPA and oxoDTA(with different isomers being formed depending on the presence of ASA).EFAD-4 to -6 were derived from n-6 and n-9 PUFAs. However, the lowlevels and the presence of several isomers did not allow a detailedstructural characterization of these latter species.

Accordingly, the inducible enzyme COX-2 was required for EFADsbiosynthesis although we cannot exclude the possibility that additionalmechanisms may be involved in their formation. In fact, autoxidation ofDHA to OH-DHA and the resulting formation of 10 positional isomers wasreported early in the 1980s. LOXs (i.e. 5-LOX and in some cases 12-LOXand 15-LOX) can initiate the oxidation of PUFAs as well. Finally,cytochrome p450 (CYP) monooxygenases have been reported to catalyze theNADPH-dependent oxidation of PUFAs and CYP4F8 has been shown to catalyzethe hydroxylation of AA and DPA (22:5n-6) mainly at the n-3 position.While the formation of hydroxy-derivatives of PUFAs has already beendescribed, further oxidation to the corresponding oxo-species has onlybeen observed for hydroxy-ETA. Moreover, despite the knowledge on(6E,8Z,11Z,14Z)-5-oxoicosa-6,8,11,14-tetranoic acid (5-oxoETE) and KODE,there is a lack of research on similar 22-carbon species. The oxidationof hydroxyl groups on bioactive lipids has been generally viewed as astep in metabolic inactivation, but we propose that such a reaction mayinstead confer novel beneficial biologic activity. Here we report abifurcation at the point where hydroxy-derivatives of n-3 PUFAs could befurther oxidized by LOXs to Rvs and neuroprotectins. We show thatmonohydroxy-PUFA derivatives are also converted to the correspondingcarbonyl species generating bioactive electrophilic lipids.

Several dehydrogenase enzymes have already been described that could beinvolved in the second oxidation step of EFAD formation. For example,the enzyme 15-hydroxyprostaglandin dehydrogenase is a candidate for thisreaction since it has been reported to catalyze the formation of15-oxoETE and the oxidation of Resolvins D1 and E1 at position −17 MolPharmacol. 2009 Jun. 17. [Epub ahead of print]). Similarly, the LTB₄12-hydroxy dehydrogenase/prostaglandin reductase (LTB₄12-HD/PGR)catalyzes the NADP⁺-dependent reduction of hydroxy-eicosanoids to thecorresponding α,β-unsaturated oxo-derivatives. In the case of 5-oxoETEformation, the 5-lipoxygenase product 5-hydroxyeicosatetranoic acid isfurther oxidized by 5-hydroxyeicosanoid dehydrogenase (5-HEDH) to5-oxoETE. As HEDH can catalyze the reaction of 5-HETE to 5-oxoETE inboth the forward and reverse direction, the formation of 5-oxoETE isfavored by a high NADP⁺:NADPH ratio (a condition symptomatic of cellsunder oxidative stress). It is interesting to note that while HEDHactivity is present in myeloid cells, it is most significantly inducedfollowing differentiation to macrophages using PMA.

The adduction of EFADs to proteins and to GSH demonstrated the role theyplay as potential modulators of protein function and as electrophilicsignal transducers. RES adduction to proteins, such as the covalentmodification of GAPDH by NO₂—FA, can alter protein's activity orsubcellular location. RES can also modulate gene expression bycovalently binding to transcriptional regulators, as exemplified byNO₂FA and 15d-PGJ₂ adduction to the p65 subunit of NFκB, thus preventingDNA binding. In other cases, RES form covalent adducts with proteinsthat associate with transcription factors (e.g. 15d-PGJ₂ adduction tothe Nrf2 inhibitor Keap1). Moreover, RES participate in signaling byforming covalent adducts with GSH. Approximately 50% of the EFADsrecovered from activated RAW264.7 cell lysate were adducted to protein(FIG. 9 a ), but this value did not include EFADs that were bound tosmall molecules such as GSH. Both intracellular and extracellular(secreted) GS-EFAD-2 (and GS-EFAD-1) adducts were identified byRP-HPLC-MS/MS. Interestingly, while both GS-13-oxoDPA and GS-17-oxoDPAadducts were detected intracellularly for RAW264.7 cells, only theGS-13-oxoDPA adduct was detected extracellularly. This observation maybe due to several possibilities; treatment of RAW264.7 cells with ASAmay affect the secretory pathway, GS-17-oxoDPA may not be secreted asefficiently as GS-13-oxoDPA, or GS-17-oxoDPA may be further metabolizedmore rapidly than GS-13-oxoDPA once secreted.

In addition to GSH and GAPDH-adduct formation, the modulation of severalsignaling pathways by EFADs confirmed their role as endogenouslyproduced anti-inflammatory signaling mediators. According to theirelectrophilic nature, 17-oxoDHA and 17-oxoDPA induced the anti-oxidantresponse by promoting nuclear accumulation of Nrf2 and the expression oftwo major Nrf2 target genes, HO-1 and Nqo-1. The 17-oxo-standards alsoacted as agonists of PPARγ, suggesting that EFADs may exert someanti-inflammatory effects through PPARγ activation. This was consistentwith previous observations that activation of PPARγ by lowconcentrations of the synthetic ligand Rosiglitazone inhibits theexpression of a small set of IFNγ and LPS-dependent genes in primarymouse macrophages. Additionally, 17-oxoDPA and 17-oxoDHA inhibited IFNγand LPS-induced cytokine production in a dose-dependent manner inRAW264.7 cells. Further evidence concerning the anti-inflammatorysignaling properties of EFADs was the dose-dependent inhibition of iNOSexpression and activity by 17-oxoDPA and 17-oxoDHA following macrophageactivation with IFNγ and Kdo₂. Surprisingly, EFAD-1 and -2 did notaffect NF-κB DNA binding activity, p65 nuclear translocation, or STAT-1phosphorylation (data not shown) in RAW264.7 suggesting that theinhibition of cytokine and iNOS expression was independent of thesesignaling pathways. Interestingly, COX-2 induction in response to Kdo₂and IFNγ was not affected by EFAD treatment. Overall these findingssuggest that EFADs may exert their anti-inflammatory actions viapathways other than NF-κB and STAT-1. The activation of PPARγ may be apossibility especially because the activation of PPARγ differentiallyaffects iNOS and COX-2 expression and can generate a pattern of cytokineexpression similar to what we have observed without affecting NF-κBactivation. Additional evidence supporting a role for EFADs as signalingmediators was the observation that 17-oxoDPA and 17-oxoDHA covalentlybind Cys and His residues in GAPDH, giving a similar pattern to thatpreviously observed for NO₂—FA. Finally, preliminary data indicate thatEFAD-1 and 2- may promote cytoprotective effects via the activation ofthe heat shock response, possibly by inducing activation of thetranscription factor Hsf1 and the subsequent transcription of targetgenes, such as Hsp70 and Hsp40. This would represent a further mechanismthrough which EFADs may exert their beneficial actions. Overall, whilerecognized signaling pathways that are modulated by electrophiles weretested, it is probable that EFADs each have their own unique signalingprofiles and receptors. Further investigation is currently underway toelucidate these profiles.

The potential of the present discovery can be fully appreciated whenconsidering EFADs biological properties as a whole: they are beneficialbioactive lipids derived from omega-3 fatty acids, produced via theaction of COX-2 and whose formation is enhanced by aspirin. In thisscenario, the yet-to-be fully elucidated beneficial roles of COX-2 andomega-3 fatty acids in resolution of inflammation and their crucial rolein cardiovascular homeostasis suggest that COX-2 derived EFADs maycontribute to mediating these actions. Furthermore, the ASA-dependentenhancement of EFAD biosynthesis further strengthens this hypothesissuggesting that the protective and anti-inflammatory effects of EFADsthat we observed in cellular models may participate in transducing someof the beneficial actions of omega-3 fatty acids, COX-2 and ASA in humanhealth.

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What is claimed is:
 1. A pharmaceutical formulation comprising: aneffective amount of a compound having a formula of:

wherein R₁ is hydrogen, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl,(C₁-C₄)alkoxy, (C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl,(C₁-C₈)hydroxyalkyl, (C₃-C₈)cycloalkyl, (C₄-C₈)bicycloalkyl,(C₃-C₈)heterocycloalkyl, heteroaryl, aryl,(C₃-C₈)cycloalkyl(C₁-C₆)alkyl, (C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl,heteroaryl(C₁-C₆)alkyl or aryl(C₁-C₆)alkyl; X is sulfur, oxygen, ornitrogen atom; and m is 0 to 15; and a pharmaceutically acceptablecarrier.
 2. A pharmaceutical formulation comprising: an effective amountof a compound having a formula of:

wherein R₁, R₂ and R₃ are each independently hydrogen, (C₁-C₈)alkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkoxy,(C₁-C₄)alkoxy(C₁-C₄)alkyl, (C₁-C₈)fluoroalkyl, (C₁-C₈)hydroxyalkyl,(C₃-C₈)cycloalkyl, (C₄-C₈)bicycloalkyl, (C₃-C₈)heterocycloalkyl,heteroaryl, aryl, (C₃-C₈)cycloalkyl(C₁-C₆)alkyl,(C₃-C₈)heterocycloalkyl(C₁-C₆)alkyl, heteroaryl(C₁-C₆)alkyl oraryl(C₁-C₆)alkyl; and m is 0 to 15; and a pharmaceutically acceptablecarrier.
 3. The formulation according to claim 1, wherein thepharmaceutically acceptable carrier comprises a solid, solution, powder,fluid emulsion, fluid suspension, semi-solid, or dry powder.
 4. Theformulation according to claim 1, further comprising at least onepharmaceutically acceptable diluent, filler, disintegrant, binder,lubricant, surfactant, hydrophobic vehicle, water soluble vehicle,emulsifier, buffer, humectant, moisturizer, solubilizer, antioxidant, orpreservative, or combinations thereof.
 5. The formulation according toclaim 2, wherein the pharmaceutically acceptable carrier comprises asolid, solution, powder, fluid emulsion, fluid suspension, semi-solid,or dry powder.
 6. The formulation according to claim 2, furthercomprising at least one pharmaceutically acceptable diluent, filler,disintegrant, binder, lubricant, surfactant, hydrophobic vehicle, watersoluble vehicle, emulsifier, buffer, humectant, moisturizer,solubilizer, antioxidant, or preservative, or combinations thereof.